Antigen binding molecules specific for an anti-CD19 scFv

ABSTRACT

Isolated antigen binding molecules that specifically bind to an anti-CD19 scFv comprising SEQ ID NO: 1 are provided. The antigen binding molecules can be used in the methods provided herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/717,691, filed Sep. 27, 2017, which claims the benefit of U.S.Provisional Patent Application Ser. No. 62/401,007 filed Sep. 28, 2016,the entire disclosures of which are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format, and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 26, 2017, isnamed K-103602_SL.txt and is 74,224 bytes in size.

FIELD OF THE INVENTION

This disclosure relates to antigen binding molecules, such asantibodies, which specifically bind to the anti-CD19 scFv FMC63, as wellas molecules comprising these sequences and cells presenting suchmolecules, polynucleotides encoding such antigen binding molecules, aswell as humanized forms of the antigen binding molecules; methods ofusing the antigen binding molecules are also disclosed.

BACKGROUND OF THE INVENTION

Antigen binding molecules, including antibodies, and fragments such asFabs, F(ab′)₂, scFvs, etc, are used in immunotherapy and solidphase-based applications such as biosensors, affinity chromatography,and immunoassays. These antibodies and other antigen binding moleculesgain their utility by virtue of their ability to specifically bind theirtargets.

Anti-idiotypic antibodies are a subset of antibodies, and are antibodiesraised against immunizing antibodies. These anti-idiotypic antibodiesdemonstrated specific binding against the idiotopes (unique antigenicdeterminants on the surface of the antibodies) of the immunizingantibodies. Anti-idiotypic antibodies can be generally classified intothree distinct groups: (1) antibodies are those that recognize idiotopesdistinct from the antigen-binding site (ABS) on immunizing antibodies;(2) antibodies that recognize epitopes within the ABS and mimic thestructure, and forming the so-called “internal image,” of the nominalantigen; and (3) antibodies that recognize epitopes within the ABSwithout the structural resemblance of the nominal antigen (see, e.g.,Pan et al., (1995) FASEB J 9:43-49).

FMC63 is an IgG2a mouse monoclonal antibody that recognizes CD19, whichis expressed on the surface of B cells (Zola et al., (1991) Immunol CellBiol 69:411-22). Single chain variable fragments (scFv) formed fromFMC63 comprise the targeting component of some chimeric antigenreceptors (CARs) (Kochenderfer et al., (2009) J Immunother32(7):689-702), and the scFv of FMC63 has previously been used togenerate anti-FMC63 antibodies (Jena et al., (2013) PLoS ONE8(3):e57838).

Disclosed herein are rabbit antigen binding molecules, includingantibodies, that specifically bind to the anti-CD19 scFv FMC63 (SEQ IDNO: 1), as well as molecules comprising these sequences and cellspresenting such molecules. Humanized forms of the disclosed rabbitantigen binding molecules also form as aspect of the disclosure.Applications and uses of these antigen binding molecules are alsodisclosed.

SUMMARY OF THE INVENTION

In one aspect, an isolated antigen binding molecule that specificallybinds a molecule comprising SEQ ID NO: 1 is provided. In someembodiments, the antigen binding molecule specifically binds a moleculecomprising one or more peptides (e.g., complementarity determiningregions (CDRs)) selected from the group consisting of SEQ ID NOs:74-82.In some embodiments, the antigen binding molecule is selected from thegroup consisting of an antibody, an scFv, a Fab, a Fab′, a Fv, aF(ab′)₂, a dAb, a human antibody, a humanized antibody, a chimericantibody, a monoclonal antibody, a polyclonal antibody, a recombinantantibody, an IgE antibody, an IgD antibody, an IgM antibody, an IgG1antibody, an IgG1 antibody having at least one mutation in the hingeregion, an IgG2 antibody an IgG2 antibody having at least one mutationin the hinge region, an IgG3 antibody, an IgG3 antibody having at leastone mutation in the hinge region, an IgG4 antibody, an IgG4 antibodyhaving at least one mutation in the hinge region, an antibody comprisingat least one non-naturally occurring amino acid, and any combinationthereof.

In some embodiments, the antigen binding molecule comprises a heavychain (HC) and in further embodiments the HC comprises a heavy chainvariable region (VH) sequence selected from the group consisting of SEQID NOs: 3, 15, 21, 33, 39 and 51. In still other embodiments, thevariable region (VH) of the antigen binding molecule comprises one ormore of (a) a CDR1, (b) a CDR2, and (c) a CDR3. In some embodiments, theantigen binding molecule comprises a heavy chain CDR1 selected from thegroup consisting of SEQ ID NOs: 5, 23, 41 and 53. In some embodiments,the antigen binding molecule comprises a heavy chain CDR2 selected fromthe group consisting of SEQ ID NOs: 6, 24, 42 and 54. In someembodiments the antigen binding molecule comprises a heavy chain CDR3selected from the group consisting of SEQ ID NOs: 7, 25, 43 and 55. Inadditional embodiments, the antigen binding molecule comprises a heavychain comprising a heavy chain CDR1, a heavy chain CDR2, and a heavychain CDR3, each CDR comprising an amino acid sequence shown in FIGS.5-21. In still further embodiments, an antigen binding molecule whichcomprises a VH amino acid sequence that is at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or about 100% identical to a VH of anantigen binding molecule provided herein.

In some embodiments, the antigen binding molecule comprises a lightchain (LC) and in further embodiments the LC comprises a light chainvariable region (VL) sequence selected from the group consisting of SEQID NOs: 8, 18, 27, 36, 45 and 57. In additional embodiments, thevariable region (VL) and comprises one or more of (a) a CDR1, (b) aCDR2, and (c) a CDR3. In some embodiments, the antigen binding moleculecomprises a light chain CDR1 selected from the group consisting of SEQID NOs: 11, 29, 47 and 59. In some embodiments, the antigen bindingmolecule comprises a light chain CDR2 selected from the group consistingof SEQ ID NOs: 12, 30, 48 and 60. In some embodiments, the antigenbinding molecule comprises a light chain CDR3 selected from the groupconsisting of SEQ ID NOs: 13, 31, 49 and 61. In some embodiments, thelight chain comprises a light chain CDR1, a light chain CDR2, and alight chain CDR3, each CDR comprising an amino acid sequence shown inone of FIGS. 5-21. In still further embodiments, an antigen bindingmolecule which comprises a VL amino acid sequence that is at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or about 100% identical toa VL of an antigen binding molecule provided herein.

In some embodiments, an antigen binding molecule comprises (a) a VHcomprising the amino acid sequence of SEQ ID NO: 3; and (b) a VLcomprising the amino acid sequence of SEQ ID NO: 9. In some embodiments,an antigen binding molecule comprises: (a) a VH CDR1 region comprisingthe amino acid sequence of SEQ ID NO: 5; (b) a VH CDR2 region comprisingthe amino acid sequence of SEQ ID NO: 6; (c) a VH CDR3 region comprisingthe amino acid sequence of SEQ ID NO: 7; (d) a VL CDR1 region comprisingthe amino acid sequence of SEQ ID NO: 11; (e) a VL CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 12; and (f) a VL CDR3region comprising the amino acid sequence of SEQ ID NO: 13.

In some embodiments, an antigen binding molecule comprises (a) a VHcomprising the amino acid sequence of SEQ ID NO: 15; and (b) a VLcomprising the amino acid sequence of SEQ ID NO: 18. In someembodiments, an antigen binding molecule comprises (a) a VH CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 5; (b) a VH CDR2 regioncomprising the amino acid sequence of SEQ ID NO: 6; (c) a VH CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 7; (d) a VL CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 11; (e) a VL CDR2region comprising the amino acid sequence of SEQ ID NO: 12; and (f) a VLCDR3 region comprising the amino acid sequence of SEQ ID NO: 13.

In some embodiments, an antigen binding molecule comprises (a) a VHcomprising the amino acid sequence of SEQ ID NO: 21; and (b) a VLcomprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, an antigen binding molecule comprises (a) a VH CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 23; (b) a VH CDR2region comprising the amino acid sequence of SEQ ID NO: 24; (c) a VHCDR3 region comprising the amino acid sequence of SEQ ID NO: 25; (d) aVL CDR1 region comprising the amino acid sequence of SEQ ID NO: 29; (e)a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 30;and (f) a VL CDR3 region comprising the amino acid sequence of SEQ IDNO: 31.

In some embodiments, an antigen binding molecule comprises (a) a VHcomprising the amino acid sequence of SEQ ID NO: 33; and (b) a VLcomprising the amino acid sequence of SEQ ID NO: 36. In someembodiments, an antigen binding molecule comprises (a) a VH CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 23; (b) a VH CDR2region comprising the amino acid sequence of SEQ ID NO: 24; (c) a VHCDR3 region comprising the amino acid sequence of SEQ ID NO: 25; (d) aVL CDR1 region comprising the amino acid sequence of SEQ ID NO: 29; (e)a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 30;and (f) a VL CDR3 region comprising the amino acid sequence of SEQ IDNO: 31.

In some embodiments, an antigen binding molecule comprises (a) a VHcomprising the amino acid sequence of SEQ ID NO: 39; and (b) a VLcomprising the amino acid sequence of SEQ ID NO: 45. In someembodiments, an antigen binding molecule comprises (a) a VH CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 41; (b) a VH CDR2region comprising the amino acid sequence of SEQ ID NO: 42; (c) a VHCDR3 region comprising the amino acid sequence of SEQ ID NO: 43; (d) VLCDR1 region comprising the amino acid sequence of SEQ ID NO: 47; (e) aVL CDR2 region comprising the amino acid sequence of SEQ ID NO: 48; and(f) a VL CDR3 region comprising the amino acid sequence of SEQ ID NO:49.

In a specific embodiment, an antigen binding molecule comprises (a) a VHcomprising the amino acid sequence of SEQ ID NO: 51; and (b) a VLcomprising the amino acid sequence of SEQ ID NO: 57. In someembodiments, an antigen binding molecule comprises (a) a VH CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 53; (b) a VH CDR2region comprising the amino acid sequence of SEQ ID NO: 54; (c) a VHCDR3 region comprising the amino acid sequence of SEQ ID NO: 55; (d) aVL CDR1 region comprising the amino acid sequence of SEQ ID NO: 59; (e)a VL CDR2 region comprising the amino acid sequence of SEQ ID NO: 60;and (f) a VL CDR3 region comprising the amino acid sequence of SEQ IDNO: 61.

In various embodiments, an antigen binding molecule provided hereinfurther comprises a detectable label, and can be selected from the groupconsisting of a fluorescent label, a photochromic compound, aproteinaceous fluorescent label, a magnetic label, a radiolabel, and ahapten. In various embodiments, a fluorescent label is selected from thegroup consisting of an Atto dye, an Alexafluor dye, quantum dots,Hydroxycoumarin, Aminocouramin, Methoxycourmarin, Cascade Blue, PacificBlue, Pacific Orange Lucifer Yellow, NBD, R-Phycoerythrin (PE), PE-Cy5conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX,Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC,X-Rhodamine, Lissamine Rhocamine B, Texas Red, Allophycocyanin (APC),APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66Hmutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire,Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation),mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan,Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65Lmutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1,TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1,Kusabira Orange, mOrange, Allophycocyanin (APC), mKO, TurboRFP,tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

In another aspect, a composition comprising an antigen binding moleculedisclosed herein is provided. Also provided is a polynucleotide encodingthe heavy chain of an antigen binding molecule disclosed herein, and apolynucleotide encoding the light chain of an antigen binding moleculedisclosed herein. A vector comprising the polynucleotides is alsodisclosed. Further, a cell comprising one or more such vectors isdisclosed. In various embodiments, the cell comprises a cell selectedfrom the group consisting of a CHO cell, a Sp2/0 cell, a rabbit cell andan E. coli cell. A method of making an antigen binding moleculedisclosed herein comprising incubating a cell disclosed herein undersuitable conditions is provided.

In another aspect, a method of administering a dose of a medicament to asubject, the dose comprising a preselected number of cells presenting atherapeutic molecule comprising SEQ ID NO: 1, is provided. In someembodiments the method comprises (a) providing a sample of known volumecomprising a population comprising a known number of cells, which cellsare known or suspected to be presenting a molecule comprising SEQ ID NO:1; (b) providing an aliquot of the sample comprising a population ofcells presenting a therapeutic molecule comprising SEQ ID NO: 1; (c)providing an antigen binding molecule that specifically binds the SEQ IDNO: 1, the antigen binding molecule further comprising a detectablelabel; (d) contacting the aliquot of (b) with the antigen bindingmolecule of (c) under conditions that permit the formation of a bindingcomplex comprising a cell present in the sample and the antigen bindingmolecule; (e) determining the fraction of cells present in a bindingcomplex of (d) in the aliquot; (f) determining the concentration ofcells presenting a molecule comprising SEQ ID NO: 1 in the sample, basedon the fraction of cells determined in (e); (g) determining the volumeof the sample that comprises the selected number of cells; and (h)administering the volume of the sample determined in (g) to the subject.

In some embodiments of the method, (a) the molecule comprising SEQ IDNO: 1 is a CAR; and (b) the cell is an immune cell selected from thegroup consisting of CD8+ T cells, CD4+ T cells, tumor infiltratinglymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, andNK-T cells. In another embodiment, the CAR further comprises a molecule,or a fragment thereof, selected from the group consisting of CD28,OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1(PD-1), inducible T cell co-stimulator (ICOS), lymphocytefunction-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT, (TNFSF14),NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1molecule, TNF receptor proteins, an Immunoglobulin protein, cytokinereceptor, integrins, Signaling Lymphocytic Activation Molecules (SLAMproteins), activating NK cell receptors, BTLA, a Toll ligand receptor,ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAH-R, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103,ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In an additional embodiment, the detectable labelis selected from the group consisting of a fluorescent label, aphotochromic compound, a proteinaceous fluorescent label, a magneticlabel, a radiolabel, and a hapten. In further embodiments, thefluorescent label is selected from the group consisting of an Atto dye,an Alexafluor dye, quantum dots, Hydroxycoumarin, Aminocouramin,Methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange LuciferYellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates,Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhocamine B, TexasRed, Allophycocyanin (APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4,DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2,Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet,GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65Amutation), Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP,TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, AzamiGreen, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP,ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO,TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. In additionalembodiments, the immune cell is a T cell and in still furtherembodiments the T cell is disposed in vitro or the T cell is disposed invivo. In other embodiments, the T cell is in one of blood, extractedtissue, tissue grown ex vivo, and cell culture media. In someembodiments, the T cell is an autologous T cell, and in otherembodiments the T cell is an allogenic T cell. In an embodiment, thedose is 1.0×10⁶ cells per kilogram of the subject. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

In another aspect, a method of administering a dose of a medicament to asubject, the dose comprising a preselected number of cells presenting atherapeutic molecule comprising CDR sequences according to any one ofSEQ ID Nos: 74-82, is provided. In some embodiments, the methodcomprises (a) providing a sample of known volume comprising a populationcomprising a known number of cells, which cells are known or suspectedto be presenting a molecule comprising CDR sequences according to anyone of SEQ ID Nos: 74-82; (b) providing an aliquot of the samplecomprising a population of cells presenting a therapeutic moleculecomprising CDR sequences according to any one of SEQ ID Nos: 74-82; (c)providing an antigen binding molecule that specifically binds a moleculecomprising CDR sequences according to any one of SEQ ID Nos: 74-82, theantigen binding molecule further comprising a detectable label; (d)contacting the aliquot of (b) with the antigen binding molecule of (c)under conditions that permit the formation of a binding complexcomprising a cell present in the sample and the antigen bindingmolecule; (e) determining the fraction of cells present in a bindingcomplex of (d) in the aliquot; (f) determining the concentration ofcells presenting a molecule comprising CDR sequences according to anyone of SEQ ID Nos: 74-82 in the sample, based on the fraction of cellsdetermined in (e); (g) determining the volume of the sample thatcomprises the selected number of cells; and (h) administering the volumeof the sample determined in (g) to the subject.

In some embodiments of the method, (a) the molecule comprising CDRsequences according to any one of SEQ ID Nos: 74-82 is a CAR; and (b)the cell is an immune cell selected from the group consisting of CD8+ Tcells, CD4+ T cells, tumor infiltrating lymphocytes (TILs), NK cells,TCR-expressing cells, dendritic cells, and NK-T cells. In someembodiments, the CAR further comprises a molecule, or a fragmentthereof, selected from the group consisting of CD28, OX-40, 4-1BB/CD137,CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cellco-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1,CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276(B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gammareceptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulinprotein, cytokine receptor, integrins, Signaling Lymphocytic ActivationMolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE,CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In some embodiments, the detectable label isselected from the group consisting of a fluorescent label, aphotochromic compound, a proteinaceous fluorescent label, a magneticlabel, a radiolabel, and a hapten. In further embodiments, thefluorescent label is selected from the group consisting of an Atto dye,an Alexafluor dye, quantum dots, Hydroxycoumarin, Aminocouramin,Methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange LuciferYellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates,Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhocamine B, TexasRed, Allophycocyanin (APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4,DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2,Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet,GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65Amutation), Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP,TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, AzamiGreen, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP,ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO,TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. In additionalembodiments, the immune cell is a T cell and in still furtherembodiments the T cell is disposed in vitro or the T cell is disposed invivo. In other embodiments, the T cell is in one of blood, extractedtissue, tissue grown ex vivo, and cell culture media. In someembodiments, the T cell is an autologous T cell, and in otherembodiments the T cell is an allogenic T cell. In an embodiment, thedose is 1.0×10⁶ cells per kilogram of the subject. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

In another aspect, a method of determining a number of cells presentinga molecule comprising SEQ ID NO: 1 in a sample is provided. In anembodiment, the method comprises (a) providing a sample comprising cellsknown or suspected to be presenting a molecule comprising SEQ ID NO: 1;(b) contacting the sample of (a) with an antigen binding molecule thatspecifically binds the molecule comprising SEQ ID NO: 1, the antigenbinding molecule further comprising a detectable label, under conditionsthat permit the formation of a binding complex comprising a cell presentin the sample and the antigen binding molecule; and (c) determining thenumber of cells present in a binding complex of (b) in the sample.

In some embodiments of the disclosed method, (a) the molecule comprisingSEQ ID NO: 1 is a CAR; and (b) the cell is an immune cell selected fromthe group consisting of CD8+ T cells, CD4+ T cells, tumor infiltratinglymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, andNK-T cells. In some embodiments, the CAR further comprises a molecule,or a fragment thereof, selected from the group consisting of CD28,OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1(PD-1), inducible T cell co-stimulator (ICOS), lymphocytefunction-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT, (TNFSF14),NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1molecule, TNF receptor proteins, an Immunoglobulin protein, cytokinereceptor, integrins, Signaling Lymphocytic Activation Molecules (SLAMproteins), activating NK cell receptors, BTLA, a Toll ligand receptor,ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103,ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In some embodiments, the detectable label isselected from the group consisting of a fluorescent label, aphotochromic compound, a proteinaceous fluorescent label, a magneticlabel, a radiolabel, and a hapten. In some embodiments, the fluorescentlabel is selected from the group consisting of an Atto dye, anAlexafluor dye, quantum dots, Hydroxycoumarin, Aminocouramin,Methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange LuciferYellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates,Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhocamine B, TexasRed, Allophycocyanin (APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4,DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2,Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet,GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65Amutation), Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP,TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, AzamiGreen, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP,ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO,TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. In additionalembodiments, the immune cell is a T cell and in still furtherembodiments the T cell is disposed in vitro or the T cell is disposed invivo. In other embodiments, the T cell is in one of blood, extractedtissue, tissue grown ex vivo, and cell culture media. In someembodiments, the T cell is an autologous T cell, and in otherembodiments the T cell is an allogenic T cell. Additionally, in variousembodiments of the disclosed method, the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

In another aspect, a method of determining a number of cells presentinga molecule comprising CDR sequences according to any one of SEQ ID Nos:74-82 in a sample is provided. In some embodiments, the method comprises(a) providing a sample comprising cells known or suspected to bepresenting a molecule comprising CDR sequences according to any one ofSEQ ID Nos: 74-82; (b) contacting the sample of (a) with an antigenbinding molecule that specifically binds the molecule comprising CDRsequences according to any one of SEQ ID Nos: 74-82 is provided, theantigen binding molecule further comprising a detectable label, underconditions that permit the formation of a binding complex comprising acell present in the sample and the antigen binding molecule; and (c)determining the number of cells present in a binding complex of (b) inthe sample.

In some embodiments of the disclosed method, (a) the molecule comprisingCDR sequences according to any one of SEQ ID Nos: 74-82 is a CAR; and(b) the cell is an immune cell selected from the group consisting ofCD8+ T cells, CD4+ T cells, tumor infiltrating lymphocytes (TILs), NKcells, TCR-expressing cells, dendritic cells, and NK-T cells. In someembodiments, the CAR further comprises a molecule, or a fragmentthereof, selected from the group consisting of CD28, OX-40, 4-1BB/CD137,CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cellco-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1,CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276(B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gammareceptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulinprotein, cytokine receptor, integrins, Signaling Lymphocytic ActivationMolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE,CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In some embodiments, the detectable label isselected from the group consisting of a fluorescent label, aphotochromic compound, a proteinaceous fluorescent label, a magneticlabel, a radiolabel, and a hapten. In some embodiments, the fluorescentlabel is selected from the group consisting of an Atto dye, anAlexafluor dye, quantum dots, Hydroxycoumarin, Aminocouramin,Methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange LuciferYellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates,Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhocamine B, TexasRed, Allophycocyanin (APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4,DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2,Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet,GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65Amutation), Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP,TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, AzamiGreen, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP,ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO,TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. In additionalembodiments, the immune cell is a T cell and in still furtherembodiments the T cell is disposed in vitro or the T cell is disposed invivo. In other embodiments, the T cell is in one of blood, extractedtissue, tissue grown ex vivo, and cell culture media. In someembodiments, the T cell is an autologous T cell, and in otherembodiments the T cell is an allogenic T cell. Additionally, in variousembodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

In another aspect, a method of isolating a molecule comprising SEQ IDNO: 1, is provided. In an embodiment, the method comprises (a) providinga sample known or suspected to comprise a molecule comprising SEQ ID NO:1; (b) providing an antigen binding molecule that specifically binds amolecule comprising SEQ ID NO: 1, optionally comprising a detectablelabel; (c) contacting the sample with the antigen binding molecule,under conditions that permit the formation of a binding complexcomprising the molecule comprising SEQ ID NO: 1 and the antigen bindingmolecule; (d) separating any molecules not part of a binding complexfrom formed binding complexes; and (e) separating a formed bindingcomplex into: (a) a molecule comprising SEQ ID NO: 1, and (b) an antigenbinding molecule.

In embodiments of the disclosed method, the molecule comprising SEQ IDNO: 1 is a CAR. In embodiments, the CAR further comprises a molecule, ora fragment thereof, selected from the group consisting of CD28, OX-40,4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1),inducible T cell co-stimulator (ICOS), lymphocyte function-associatedantigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3zeta, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a),DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins,an Immunoglobulin protein, cytokine receptor, integrins, SignalingLymphocytic Activation Molecules (SLAM proteins), activating NK cellreceptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1,GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma,IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX,CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand thatspecifically binds with CD83, and combinations thereof. In someembodiments, the antigen binding molecule is disposed on a surfaceselected from the group consisting of an agarose bead, a magnetic bead,a plastic welled plate, a glass welled plate, a ceramic welled plate anda cell culture bag. some embodiments, the detectable label is selectedfrom the group consisting of a fluorescent label, a photochromiccompound, a proteinaceous fluorescent label, a magnetic label, aradiolabel, and a hapten. In some embodiments, the fluorescent label isselected from the group consisting of an Atto dye, an Alexafluor dye,quantum dots, Hydroxycoumarin, Aminocouramin, Methoxycourmarin, CascadeBlue, Pacific Blue, Pacific Orange Lucifer Yellow, NBD, R-Phycoerythrin(PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed,FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7,TRITC, X-Rhodamine, Lissamine Rhocamine B, Texas Red, Allophycocyanin(APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP(Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv,T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66Wmutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation),Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP,GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green,ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP,ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO,TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

In another aspect, a method of isolating a molecule comprising CDRsequences according to any one of SEQ ID Nos: 74-82, is provided. In anembodiment, the method comprises (a) providing a sample known orsuspected to comprise a molecule comprising CDR sequences according toany one of SEQ ID Nos: 74-82; (b) providing an antigen binding moleculethat specifically binds a molecule comprising CDR sequences according toany one of SEQ ID Nos: 74-82, optionally comprising a detectable label;(c) contacting the sample with the antigen binding molecule, underconditions that permit the formation of a binding complex comprising themolecule comprising CDR sequences according to any one of SEQ ID Nos:74-82 and the antigen binding molecule; (d) separating any molecules notpart of a binding complex from formed binding complexes; and (e)separating a formed binding complex into: (a) a molecule comprising CDRsequences according to any one of SEQ ID Nos: 74-82, and (b) an antigenbinding molecule.

In some embodiments of the disclosed method, the molecule comprising CDRsequences according to any one of SEQ ID Nos: 74-82 is a CAR. Inembodiments, the CAR further comprises a molecule, or a fragmentthereof, selected from the group consisting of CD28, OX-40, 4-1BB/CD137,CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cellco-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1,CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276(B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gammareceptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulinprotein, cytokine receptor, integrins, Signaling Lymphocytic ActivationMolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAH-R, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE,CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In some embodiments, the antigen binding moleculeis disposed on a surface selected from the group consisting of anagarose bead, a magnetic bead, a plastic welled plate, a glass welledplate, a ceramic welled plate and a cell culture bag. In someembodiments, the detectable label is selected from the group consistingof a fluorescent label, a photochromic compound, a proteinaceousfluorescent label, a magnetic label, a radiolabel, and a hapten. In someembodiments, the fluorescent label is selected from the group consistingof an Atto dye, an Alexafluor dye, quantum dots, Hydroxycoumarin,Aminocouramin, Methoxycourmarin, Cascade Blue, Pacific Blue, PacificOrange Lucifer Yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates,PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein,BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine,Lissamine Rhocamine B, Texas Red, Allophycocyanin (APC), APC-Cy7conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66Hmutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire,Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation),mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan,Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65Lmutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1,TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1,Kusabira Orange, mOrange, Allophycocyanin (APC), mKO, TurboRFP,tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

In another aspect, a method of determining the presence or absence of amolecule comprising SEQ ID NO: 1 in a sample. In some embodiments, themethod comprises (a) providing a sample known or suspected to comprise amolecule comprising SEQ ID NO: 1; (b) providing an antigen bindingmolecule comprising a detectable label that specifically binds amolecule comprising SEQ ID NO: 1; (c) contacting the sample with theantigen binding molecule under conditions that permit the formation of abinding complex; (d) separating any molecules not part of a bindingcomplex from formed binding complexes; and (e) detecting the presence orabsence of a binding complex.

In some embodiments, the molecule comprising SEQ ID NO: 1 is a CAR, andin further embodiments, the CAR further comprises a molecule, or afragment thereof, selected from the group consisting of CD28, OX-40,4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1),inducible T cell co-stimulator (ICOS), lymphocyte function-associatedantigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3zeta, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a),DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins,an Immunoglobulin protein, cytokine receptor, integrins, SignalingLymphocytic Activation Molecules (SLAM proteins), activating NK cellreceptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1,GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma,IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX,CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand thatspecifically binds with CD83, and combinations thereof. In additionalembodiments, the antigen binding molecule is disposed on a surfaceselected from the group consisting of an agarose bead, a magnetic bead,a plastic welled plate, a glass welled plate, a ceramic welled plate anda cell culture bag. In further embodiments, the detectable label isselected from the group consisting of a fluorescent label, aphotochromic compound, a proteinaceous fluorescent label, a magneticlabel, a radiolabel, and a hapten. In still further embodiments, thefluorescent label is selected from the group consisting of an Atto dye,an Alexafluor dye, quantum dots, Hydroxycoumarin, Aminocouramin,Methoxycourmarin, Cascade Blue, Pacific Blue, Pacific Orange LuciferYellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates,Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhocamine B, TexasRed, Allophycocyanin (APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4,DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2,Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet,GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65Amutation), Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP,TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, AzamiGreen, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP,ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO,TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

In another aspect, a method of determining the presence or absence of amolecule comprising CDR sequences according to any one of SEQ ID Nos:74-82 in a sample. In some embodiments, the method comprises (a)providing a sample known or suspected to comprise a molecule comprisingCDR sequences according to any one of SEQ ID Nos: 74-82; (b) providingan antigen binding molecule comprising a detectable label thatspecifically binds a molecule comprising CDR sequences according to anyone of SEQ ID Nos: 74-82; (c) contacting the sample with the antigenbinding molecule under conditions that permit the formation of a bindingcomplex; (d) separating any molecules not part of a binding complex fromformed binding complexes; and (e) detecting the presence or absence of abinding complex.

In some embodiments, the molecule comprising CDR sequences according toany one of SEQ ID Nos: 74-82 is a CAR, and in further embodiments, theCAR further comprises a molecule, or a fragment thereof, selected fromthe group consisting of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30,CD40, Programmed Death-1 (PD-1), inducible T cell co-stimulator (ICOS),lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT,(TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokinereceptor, integrins, Signaling Lymphocytic Activation Molecules (SLAMproteins), activating NK cell receptors, BTLA, a Toll ligand receptor,ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103,ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In additional embodiments, the antigen bindingmolecule is disposed on a surface selected from the group consisting ofan agarose bead, a magnetic bead, a plastic welled plate, a glass welledplate, a ceramic welled plate and a cell culture bag. In furtherembodiments, the detectable label is selected from the group consistingof a fluorescent label, a photochromic compound, a proteinaceousfluorescent label, a magnetic label, a radiolabel, and a hapten. Instill further embodiments, the fluorescent label is selected from thegroup consisting of an Atto dye, an Alexafluor dye, quantum dots,Hydroxycoumarin, Aminocouramin, Methoxycourmarin, Cascade Blue, PacificBlue, Pacific Orange Lucifer Yellow, NBD, R-Phycoerythrin (PE), PE-Cy5conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX,Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC,X-Rhodamine, Lissamine Rhocamine B, Texas Red, Allophycocyanin (APC),APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66Hmutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire,Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation),mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan,Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65Lmutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1,TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1,Kusabira Orange, mOrange, Allophycocyanin (APC), mKO, TurboRFP,tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

In yet another aspect, a method of increasing the concentration of cellspresenting a molecule comprising SEQ ID NO: 1 is provided. In someembodiments, the method comprises (a) providing a sample comprising acell known or suspected to present a molecule comprising SEQ ID NO: 1;(b) providing an antigen binding molecule that specifically binds amolecule comprising SEQ ID NO: 1, optionally comprising a detectablelabel; (c) contacting the sample with the antigen binding molecule underconditions that permit the formation of a binding complex comprising themolecule comprising SEQ ID NO: 1 and the antigen binding molecule; (d)removing any components not part of a binding complex; and (e) repeatingsteps (a)-(d) a desired number of times.

In an embodiment, (a) the molecule comprising SEQ ID NO: 1 is a CAR; and(b) the cell is an immune cell selected from the group consisting ofCD8+ T cells, CD4+ T cells, tumor infiltrating lymphocytes (TILs), NKcells, TCR-expressing cells, dendritic cells, and NK-T cells. In otherembodiments, the CAR further comprises a molecule, or a fragmentthereof, selected from the group consisting of CD28, OX-40, 4-1BB/CD137,CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cellco-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1,CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276(B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gammareceptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulinprotein, cytokine receptor, integrins, Signaling Lymphocytic ActivationMolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE,CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In additional embodiments, the immune cell is a Tcell and in still further embodiments the T cell is disposed in vitro orthe T cell is disposed in vivo. In other embodiments, the T cell is inone of blood, extracted tissue, tissue grown ex vivo, and cell culturemedia. In some embodiments, the T cell is an autologous T cell, and inother embodiments the T cell is an allogenic T cell. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof. In further embodiments, the detectable label is selectedfrom the group consisting of a fluorescent label, a photochromiccompound, a proteinaceous fluorescent label, a magnetic label, aradiolabel, and a hapten. In embodiments, the fluorescent label isselected from the group consisting of an Atto dye, an Alexafluor dye,quantum dots, Hydroxycoumarin, Aminocouramin, Methoxycourmarin, CascadeBlue, Pacific Blue, Pacific Orange Lucifer Yellow, NBD, R-Phycoerythrin(PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed,FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7,TRITC, X-Rhodamine, Lissamine Rhocamine B, Texas Red, Allophycocyanin(APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP(Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv,T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66Wmutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation),Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP,GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green,ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP,ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO,TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

In yet another aspect, a method of increasing the concentration of cellspresenting a molecule comprising CDR sequences according to any one ofSEQ ID Nos: 74-82 is provided. In an embodiment, the method comprises(a) providing a sample comprising a cell known or suspected to present amolecule comprising CDR sequences according to any one of SEQ ID Nos:74-82; (b) providing an antigen binding molecule that specifically bindsa molecule comprising CDR sequences according to any one of SEQ ID Nos:74-82, optionally comprising a detectable label; (c) contacting thesample with the antigen binding molecule under conditions that permitthe formation of a binding complex comprising the molecule comprisingCDR sequences according to any one of SEQ ID Nos: 74-82 and the antigenbinding molecule; (d) removing any components not part of a bindingcomplex; and (e) repeating steps (a)-(d) a desired number of times.

In an embodiment, (a) the molecule comprising CDR sequences according toany one of SEQ ID Nos: 74-82 is a CAR; and (b) the cell is an immunecell selected from the group consisting of CD8+ T cells, CD4+ T cells,tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells,dendritic cells, and NK-T cells. In other embodiments, the CAR furthercomprises a molecule, or a fragment thereof, selected from the groupconsisting of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40,Programmed Death-1 (PD-1), inducible T cell co-stimulator (ICOS),lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT,(TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokinereceptor, integrins, Signaling Lymphocytic Activation Molecules (SLAMproteins), activating NK cell receptors, BTLA, a Toll ligand receptor,ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAH-R, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103,ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In additional embodiments, the immune cell is a Tcell and in still further embodiments the T cell is disposed in vitro orthe T cell is disposed in vivo. In other embodiments, the T cell is inone of blood, extracted tissue, tissue grown ex vivo, and cell culturemedia. In some embodiments, the T cell is an autologous T cell, and inother embodiments the T cell is an allogenic T cell. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof. In further embodiments, the detectable label is selectedfrom the group consisting of a fluorescent label, a photochromiccompound, a proteinaceous fluorescent label, a magnetic label, aradiolabel, and a hapten. In embodiments, the fluorescent label isselected from the group consisting of an Atto dye, an Alexafluor dye,quantum dots, Hydroxycoumarin, Aminocouramin, Methoxycourmarin, CascadeBlue, Pacific Blue, Pacific Orange Lucifer Yellow, NBD, R-Phycoerythrin(PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed,FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7,TRITC, X-Rhodamine, Lissamine Rhocamine B, Texas Red, Allophycocyanin(APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP(Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv,T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66Wmutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation),Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP,GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green,ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP,ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO,TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

In another aspect, a method of depleting a population of immune cellspresenting a molecule comprising SEQ ID NO: 1 is provided. In someembodiments, the method comprises (a) providing a population of immunecells to be depleted, wherein the immune cells are known or suspected tobe presenting a molecule comprising SEQ ID NO: 1; and (b) contacting theimmune cells with an antigen binding molecule that specifically binds to(a) the molecule comprising SEQ ID NO: 1, and (b) an activating moleculeexpressed on the surface of the an immune cell not presenting themolecule comprising SEQ ID NO:1, under conditions that permit theformation of a ternary binding complex comprising the moleculecomprising SEQ ID NO: 1, the activating molecule and the antigen bindingmolecule. In further embodiments, (a) the molecule comprising SEQ ID NO:1 is a CAR; and (b) the immune cell selected from the group consistingof CD8+ T cells, CD4+ T cells, tumor infiltrating lymphocytes (TILs), NKcells, TCR-expressing cells, dendritic cells, and NK-T cells. In stillfurther embodiments, the CAR further comprises a molecule, or a fragmentthereof, selected from the group consisting of CD28, OX-40, 4-1BB/CD137,CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cellco-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1,CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276(B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gammareceptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulinprotein, cytokine receptor, integrins, Signaling Lymphocytic ActivationMolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE,CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In additional embodiments, the immune cell is a Tcell and in still further embodiments the T cell is disposed in vitro orthe T cell is disposed in vivo. In other embodiments, the T cell is inone of blood, extracted tissue, tissue grown ex vivo, and cell culturemedia. In some embodiments, the T cell is an autologous T cell, and inother embodiments the T cell is an allogenic T cell. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

In another aspect, a method of depleting a population of immune cellspresenting a molecule comprising CDR sequences according to any one ofSEQ ID Nos: 74-82 is provided. In some embodiments, the method comprises(a) providing a population of immune cells to be depleted, wherein theimmune cells are known or suspected to be presenting a moleculecomprising CDR sequences according to any one of SEQ ID Nos: 74-82; and(b) contacting the immune cells with an antigen binding molecule thatspecifically binds to (a) the molecule comprising CDR sequencesaccording to any one of SEQ ID Nos: 74-82, and (b) an activatingmolecule expressed on the surface of the an immune cell not presentingthe molecule comprising CDR sequences according to any one of SEQ IDNos: 74-82, under conditions that permit the formation of a ternarybinding complex comprising the molecule comprising CDR sequencesaccording to any one of SEQ ID Nos: 74-82, the activating molecule andthe antigen binding molecule.

In further embodiments, (a) the molecule comprising CDR sequencesaccording to any one of SEQ ID Nos: 74-82 is a CAR; and (b) the immunecell selected from the group consisting of CD8+ T cells, CD4+ T cells,tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells,dendritic cells, and NK-T cells. In still further embodiments, the CARfurther comprises a molecule, or a fragment thereof, selected from thegroup consisting of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30,CD40, Programmed Death-1 (PD-1), inducible T cell co-stimulator (ICOS),lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT,(TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokinereceptor, integrins, Signaling Lymphocytic Activation Molecules (SLAMproteins), activating NK cell receptors, BTLA, a Toll ligand receptor,ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103,ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof. In additional embodiments, the immune cell is a Tcell and in still further embodiments the T cell is disposed in vitro orthe T cell is disposed in vivo. In other embodiments, the T cell is inone of blood, extracted tissue, tissue grown ex vivo, and cell culturemedia. In some embodiments, the T cell is an autologous T cell, and inother embodiments the T cell is an allogenic T cell. Additionally, invarious embodiments of the disclosed method the antigen binding moleculecomprises an antigen binding molecule disclosed herein, and humanizedforms thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are a series of plots showing the results of flowcytometry experiments performed using untransduced cells (FIG. 1A) andcells transduced with a construct encoding a CAR comprising theanti-CD19 scFv FMC63 (FIG. 1B) and then contacted with anti-scFvantibodies corresponding to three different parental clones (Clone 14,Clone 15 and Clone 17); the plots demonstrate specific binding of theantibodies to the expressed CAR.

FIG. 2 is a series of plots showing the results of flow cytometryexperiments performed using cells transduced with a construct encoding aCAR comprising the anti-CD19 scFv FMC63 and then contacted withanti-scFv antibodies corresponding to five different clones (Clone 14-1,Clone 14-7, Clone 15, Clone 17-1 and Clone 17-4); the plots demonstratespecific binding of the antibodies to the expressed CAR.

FIG. 3 is a series of images depicting the results ofimmunohistochemistry (IHC) studies performed using cells presenting aCAR comprising the anti-CD19 scFv FMC63; the left figure demonstratesthe specific binding of antibody Clone 7 to the CAR, while the rightfigure demonstrates the specific binding of antibody Clone 13 to theCAR.

FIGS. 4A and 4B are a series of photographs depicting the results ofimmunohistochemistry (IHC) studies performed using cells presenting aCAR comprising the anti-CD19 scFv FMC63; FIG. 4A demonstrates thespecific binding of antibody Clones 7 and 13 to the CAR, while FIG. 4Bdemonstrates the results for both antibodies using a PBMC control.

FIG. 5 is a series of sequences showing the coding and amino acidsequences for the VH region of Clone 7, the full length heavy chainamino acid sequence, and the heavy chain CDR1, CDR 2 and CDR 3 aminoacid sequences for this clone.

FIG. 6 is a series of sequences showing the coding and amino acidsequences for the VL region of Clone 7, the full length light chainamino acid sequence, and the light chain CDR1, CDR 2 and CDR 3 aminoacid sequences for this clone.

FIG. 7 is a series of sequences showing the coding and amino acidsequences for the VH region of Clone 13, the full length heavy chainamino acid sequence and the heavy chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIG. 8 is a series of sequences showing the coding and amino acidsequences for the VL region of Clone 13, the full length light chainamino acid sequence and the light chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIG. 9 is a series of sequences showing the coding and amino acidsequences for the VH region of Clone 14-1, the full length heavy chainamino acid sequence and the heavy chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIG. 10 is a series of sequences showing the coding and amino acidsequences for the VL region of Clone 14-1, the full length light chainamino acid sequence and the light chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIG. 11 is a series of sequences showing the coding and amino acidsequences for the VH region of Clone 14-7, the full length heavy chainamino acid sequence and the heavy chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIG. 12 is a series of sequences showing the coding and amino acidsequences for the VL region of Clone 14-7, the full length light chainamino acid sequence and the light chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIG. 13 is a series of sequences showing the coding and amino acidsequences for the VH region of Clone 15, the full length heavy chainamino acid sequence and the heavy chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIG. 14 is a series of sequences showing the coding and amino acidsequences for the VL region of Clone 15, the full length light chainamino acid sequence and the light chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIG. 15 is a series of sequences showing the coding and amino acidsequences for the VH region of Clone 17, the full length heavy chainamino acid sequence and the heavy chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIG. 16 is a series of sequences showing the coding and amino acidsequences for the VL region of Clone 17, the full length light chainamino acid sequence and the light chain CDR1, CDR 2 and CDR 3 amino acidsequences for this clone.

FIGS. 17A and 17B are alignments showing the variable heavy chainsequences of the six distinct antibodies identified (FIG. 17A), and acladding diagram (FIG. 17B) showing the relationship of the sequences toone another.

FIGS. 18A and 18B are alignments showing the variable light chainsequences of the six distinct antibodies identified (FIG. 18A), and acladding diagram (FIG. 18B) showing the relationship of the sequences toone another.

FIG. 19 is a table showing the CDR1, CDR2 and CDR3 sequences of theheavy and light chains of the six distinct antibodies identified,assigned based on the Kabat numbering scheme.

FIG. 20 is a table showing the CDR1, CDR2 and CDR3 sequences of theheavy and light chains of the six distinct antibodies identified,assigned based on the Chothia numbering scheme.

FIG. 21 is a table showing the CDR1, CDR2 and CDR3 sequences of theheavy and light chains of the six distinct antibodies identified,assigned based on the IMGT numbering scheme.

FIG. 22 shows flow cytometry plots used to determine binding of clones14-1, 15, and 17-4 to FMC63 and humanized variants thereof (SS, JS, AS,NS).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to anti-idiotypic antigen bindingmolecules, including antibodies, which specifically bind to antigenbinding molecules that specifically bind the amino acid sequence of theanti-CD19 scFv FMC63 (see, Nicholson et al., (1997) Mol Immunol34(16-17):1157-65).

The anti-CD19 scFv FMC63 has the amino acid sequence:

(SEQ ID NO: 1) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

Humanized forms of the antigen binding molecules, molecules comprisingthe anti-CD19 scFv FMC63 and cells presenting a molecule comprising theanti-CD19 scFv FMC63 are also provided. Additionally, polynucleotidesencoding the antigen binding molecules, as well as vectors comprisingthe polynucleotides, and in vitro cells comprising the polynucleotidesand vectors, are also disclosed.

Methods of using the disclosed antigen binding molecules are provided.The antigen binding molecules, polynucleotides, vectors, in vitro cellsand methods described herein can be used in a range of applications,e.g., as reagents to detect the presence of moieties comprising theanti-CD19 scFv FMC63, as well as molecules comprising this sequence andcells presenting such molecules, quantifying the amount of a moietycomprising anti-CD19 scFv FMC63, as well as molecules comprising thissequence and cells presenting such molecules, screening for moietiescomprising the anti-CD19 scFv FMC63, as well as molecules comprisingthis sequence and cells presenting such molecules, purifying moietiescomprising the anti-CD19 scFv FMC63, as well as molecules comprisingthis sequence and cells presenting such molecules, and biomarker studiesfocused on moieties comprising the anti-CD19 scFv FMC63, as well asmolecules comprising this sequence and cells presenting such molecules.Therapeutic uses are also provided, for example applications in whichthe biological activity of a moiety comprising the anti-CD19 scFv FMC63,as well as cells presenting such molecules, is modulated (enhanced orrepressed), as well as dose ranging studies related to therapeuticscomprising the anti-CD19 scFv FMC63, as well as molecules comprisingthis sequence and cells presenting such molecules, and cells presentingsuch molecules.

The antigen binding molecules (antibodies) disclosed herein weregenerated from hybridomas generated using B-cells of rabbit origin, butcan be readily humanized using standard methods known to those of skillin the art, as well as those described herein. Representative humanizedforms of the disclosed antigen binding molecules can be generated asdescribed herein.

I. Definitions

In order that the present disclosure may be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application. The headings provided herein are notlimitations of the various aspects of the disclosure, which aspectsshould be understood by reference to the specification as a whole.

It is understood that, wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Units, prefixes, and symbols used herein are provided using theirSystème International de Unites (SI) accepted form. Numeric ranges areinclusive of the numbers defining the range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, Juo, TheConcise Dictionary of Biomedicine and Molecular Biology, 2^(nd) ed.,(2001), CRC Press; The Dictionary of Cell & Molecular Biology, 5^(th)ed., (2013), Academic Press; and The Oxford Dictionary Of BiochemistryAnd Molecular Biology, Cammack et al. eds., 2^(nd) ed, (2006), OxfordUniversity Press, provide those of skill in the art with a generaldictionary for many of the terms used in this disclosure.

As used herein, the twenty conventional (e.g., naturally occurring)amino acids and their abbreviations follow conventional usage. See,e.g., Immunology—A Synthesis (2nd Edition), Golub and Green, eds.,Sinauer Assoc., Sunderland, Mass. (1991), which is incorporated hereinby reference for any purpose. Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as alpha-,alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids can also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, gamma-carboxyglutamate,epsilon-N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,sigma-N-methylarginine, and other similar amino acids and imino acids(e.g., 4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

As used herein, the term the terms “a” and “an” are used per standardconvention and mean one or more, unless context dictates otherwise.

As used herein, the term “about” refers to a value or composition thatis within an acceptable error range for the particular value orcomposition as determined by one of ordinary skill in the art, whichwill depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” can mean within one ormore than one standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 10% (i.e., ±10%). For example, about 5 mg can include anynumber between 4.5 mg and 5.5 mg. Furthermore, particularly with respectto biological systems or processes, the terms can mean up to an order ofmagnitude or up to 5-fold of a value. When particular values orcompositions are provided in the instant disclosure, unless otherwisestated, the meaning of “about” or “comprising essentially of” should beassumed to be within an acceptable error range for that particular valueor composition.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to be inclusive of the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one-tenth and one-hundredth of an integer), unlessotherwise indicated.

As used herein, the term “and/or” is to be understood as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or,” as used in aphrase such as ‘A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the term the use of the alternative (e.g., “or”) shouldbe understood to mean either one, both, or any combination thereof ofthe alternatives.

As used herein, the term “allogeneic” refers to any material derivedfrom one individual which is then introduced to another individual ofthe same species, e.g., allogeneic T cell transplantation.

As used herein, the term “antibody” (Ab) includes, without limitation, aglycoprotein immunoglobulin which binds specifically to an antigen. Ingeneral, an antibody can comprise at least two heavy (HC) chains and twolight (LC) chains interconnected by disulfide bonds, or an antigenbinding molecule thereof. Each HC chain comprises a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region. Theheavy chain constant region comprises three constant domains, CH1, CH2and CH3. Each LC chain comprises a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region comprises one constant domain, CL. The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL comprises three CDRs and four FRs, arranged from amino-terminusto carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe Abs may mediate the binding of the immunoglobulin to host tissues orfactors, including various cells of the immune system (e.g., effectorcells) and the first component of the classical complement system (C1q).The term “antibody” also encompasses an intact immunoglobulin or anantigen binding portion thereof that competes with the intact antibodyfor specific binding, unless otherwise specified. Antigen bindingportions can be produced by recombinant DNA techniques or by enzymaticor chemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),fragments including complementarity determining regions (CDRs),single-chain antibodies (scFv), chimeric antibodies, diabodies,triabodies, tetrabodies, and polypeptides that contain at least aportion of an immunoglobulin that is sufficient to confer specificantigen binding to the polypeptide.

The term “antibody” includes, both naturally occurring and non-naturallyoccurring (recombinantly-produced) antibodies, human and non-humanantibodies, monospecific antibodies, multispecific antibodies (includingbispecific antibodies), immunoglobulins, synthetic antibodies,tetrameric antibodies comprising two heavy chain and two light chainmolecules, an antibody light chain monomer, an antibody heavy chainmonomer, an antibody light chain dimer, an antibody heavy chain dimer,an antibody light chain-antibody heavy chain pair, intrabodies (see,e.g., Stocks, (2004) Drug Discovery Today 9(22):960-66), antibodyfusions (which term encompasses antibody-drug conjugates) and which aresometimes referred to herein as “antibody conjugates”), heteroconjugateantibodies, single domain antibodies, monovalent antibodies, singlechain antibodies or single-chain Fvs (scFv), camelized antibodies,affybodies, Fab fragments, F(ab′)₂ fragments, disulfide-linked Fvs(sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-anti-Id antibodies), minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”), andantigen-binding fragments thereof. In certain embodiments, antibodiesdescribed herein refer to polyclonal antibody populations.

A non-human antibody can be humanized using recombinant methods toreduce its immunogenicity in humans, as disclosed herein, with respectto antibodies that specifically bind the anti-CD19 scFv FMC63, as wellas molecules comprising this sequence and cells presenting suchmolecules. Where not expressly stated, and unless the context indicatesotherwise, the term “antibody” also includes an antigen-binding fragmentof an antigen binding molecule of any of the aforementionedimmunoglobulins, and includes a monovalent and a divalent fragment orportion, and a single chain antibody (i.e., a scFv).

In various embodiments, an antibody specifically binds the anti-CD19scFv FMC63 (SEQ ID NO: 1), as well as molecules comprising this sequenceand cells presenting such molecules. In some embodiments, the antibodyspecifically binds to a CAR (or component thereof) comprising SEQ IDNO:1, as well as molecules comprising this sequence, and cellspresenting such molecules; cells presenting SEQ ID NO:1 can, but neednot be, an immune cell, such as a T cell.

As used herein, the term “antigen” means any molecule that provokes animmune response or is capable of being bound by an antibody or otherantigen binding molecule. The immune response can involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. Those of skill in the art willreadily understand that any macromolecule, including virtually allproteins or peptides (including the anti-CD19 scFv FMC63; SEQ ID NO: 1),as well as molecules comprising this sequence and cells presenting suchmolecules), can serve as an antigen. Generally, an antigen can beendogenously expressed, i.e. expressed by genomic DNA, or it can berecombinantly expressed, or it can be chemically synthesized. In oneparticular embodiment, an antigen comprises all or a portion of theanti-CD19 scFv FMC63, as well as molecules comprising this sequence,which is optionally conjugated to an adjuvant such as keyhole limpethemocyanin (KLH), or to an Fc to facilitate screening.

As used herein, the term “antigen binding molecule” means a proteincomprising a portion that binds to an antigen or target protein and,optionally, a scaffold or framework portion that allows the antigenbinding portion to adopt a conformation that promotes binding of theantigen binding molecule to the antigen. Examples of the representativetypes of antigen binding molecules include a scFv, a human, mouse orrabbit antibody; a humanized antibody; a chimeric antibody; arecombinant antibody; a single chain antibody; a diabody; a triabody; atetrabody; a Fab fragment; a F(ab′)2 fragment; an IgD antibody; an IgEantibody; an IgM antibody; an IgG1 antibody; an IgG2 anti-body; an IgG3antibody; or an IgG4 antibody, and fragments thereof.

An antigen binding molecule can comprise, for example, an alternativeprotein scaffold or artificial scaffold with grafted complementaritydetermining regions (CDRs) or CDR derivatives. Such scaffolds include,but are not limited to, antibody-derived scaffolds comprising mutationsintroduced to, for example, stabilize the three-dimensional structure ofthe antigen binding molecule as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, e.g., Korndorferet al., 2003, Proteins: Structure, Function, and Bioinformatics,53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004).In addition, peptide antibody mimetics (“PAMs”) can be used, as well asscaffolds based on antibody mimetics utilizing various components (e.g.,fibronectin) as a scaffold. An antigen binding molecule can have, forexample, the structure of a naturally occurring immunoglobulin.

An antigen binding molecule can have one or more binding sites. If thereis more than one binding site, the binding sites can be identical to oneanother or they can be different. For example, a naturally occurringhuman immunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites,and is capable of specifically binding two different antigens (e.g., theanti-CD19 scFv FMC63 and a cell surface activator molecule).

In various embodiments, an antigen binding molecule is an antibody orfragment thereof, including one or more of the complementaritydetermining regions (CDRs) disclosed herein and shown in FIGS. 5-21,which specifically bind the anti-CD19 scFv FMC63, as well as moleculescomprising the anti-CD19 scFv FMC63, and cells presenting suchmolecules. In further embodiments, the antigen binding molecule binds toa CAR comprising the anti-CD19 scFv FMC63, as well as moleculescomprising the anti-CD19 scFv FMC63, and can be expressed on an immunecell, such as a T cell.

The term “autologous” refers to any material derived from the sameindividual to which it is later to be re-introduced. For example, theengineered autologous cell therapy (eACT™) methods described hereininvolve collection of lymphocytes from a patient, which are thenengineered to express a construct, e.g., a CAR construct, and thenadministered back to the same patient.

As used herein, the term “binding affinity” means the strength of thesum total of non-covalent interactions between a single binding site ofa molecule (e.g., an antigen binding molecule such as an antibody) andits binding partner (e.g., an antigen). Unless indicated otherwise, asused herein, “binding affinity” refers to intrinsic binding affinitywhich reflects a 1:1 interaction between members of a binding pair(e.g., antibody and antigen). The affinity of a molecule X for itspartner Y can generally be represented by the dissociation constant(K_(D)). Affinity can be measured and/or expressed in a number of waysknown in the art, including, but not limited to, equilibriumdissociation constant (K_(D)), and equilibrium association constant(K_(A)). The K_(D) is calculated from the quotient of k_(off)/k_(on),whereas K_(A) is calculated from the quotient of k_(on)/k_(off). k_(on)refers to the association rate constant of, e.g., an antibody to anantigen, and k_(off) refers to the dissociation of, e.g., an antibody toan antigen. The k_(on) and k_(off) can be determined by standardtechniques known to one of ordinary skill in the art, such as BIAcore®or KinExA or surface plasmon resonance.

As used herein, the term “complementarity determining region” or “CDR”means an amino acid sequence that contributes to antigen bindingspecificity and affinity. Framework regions can aid in maintaining theproper confirmation of the CDRs to promote binding between the antigenbinding molecule and an antigen. A number of definitions of the CDRs arecommonly in use: Kabat numbering, Chothia numbering, AbM numbering, orcontact numbering. The AbM definition is a compromise between the Kabatand Chothia systems, and is used by Oxford Molecular's AbM antibodymodelling software. Table A defines CDRs using each numbering system.The contact definition is based on an analysis of the available complexcrystal structures.

TABLE A Loop Kabat AbM Chothia Contact L1 L24--L34 L24--L34 L24--L34L30--L36 L2 L50--L56 L50--L56 L50--L56 L46--L55 L3 L89--L97 L89--L97L89--L97 L89--L96 H1 H31--H35B H26--H35B H26--H32 . . . 34 H30--H35B H1H31--H35 H26--H35 H26--H32 H30--H35 H2 H50--H65 H50--H58 H52--H56H47--H58 H3 H95--H102 H95--H102 H95--H102 H93--H101

The term “Kabat numbering” and like terms are recognized in the art andrefer to a system of numbering amino acid residues in the heavy andlight chain variable regions of an antibody, or an antigen bindingmolecule thereof. In certain aspects, the CDRs of an antibody can bedetermined according to the Kabat numbering system (see, e.g., Kabat etal. in Sequences of Proteins of immunological Interest, 5th Ed., NIHPublication 91-3242, Bethesda Md. 1991). Using the Kabat numberingsystem, CDRs within an antibody heavy chain molecule are typicallypresent at amino acid positions 31 to 35, which optionally can includeone or two additional amino acids, following 35 (referred to in theKabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabatnumbering system, CDRs within an antibody light chain molecule aretypically present at amino acid positions 24 to 34 (CDR1), amino acidpositions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). Insome embodiments, the CDRs of the antibodies described herein can bedescribed according to the Kabat numbering scheme, as shown in FIG. 6(although they can readily be construed in other numbering systems usingTable A above).

In certain aspects, the CDRs of an antibody can be determined accordingto the Chothia numbering scheme, which refers to the location ofimmunoglobulin structural loops (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817;Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No.7,709,226). Typically, when using the Kabat numbering convention, theChothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33,or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids95 to 102, while the Chothia CDR-L1 loop is present at light chain aminoacids 24 to 34, the Chothia CDR-L2 loop is present at light chain aminoacids 50 to 56, and the Chothia CDR-L3 loop is present at light chainamino acids 89 to 97. The end of the Chothia CDR-HI loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34). See Table A.In some embodiments, the CDRs of the antibodies described herein havebeen determined according to the Chothia numbering scheme, as shown inFIG. 20.

As used herein, a “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Incertain embodiments, one or more amino acid residues within a CDR(s) orwithin a framework region(s) of an antibody or antigen binding moleculeprovided herein (or fragment thereof) can be replaced with an amino acidresidue with a similar side chain.

Conservative amino acid substitutions, which are encompassed by thepresent disclosure, can encompass non-naturally occurring amino acidresidues, which are typically incorporated by chemical peptide synthesisrather than by synthesis in biological systems. These includepeptidomimetics and other reversed or inverted forms of amino acidmoieties. Naturally occurring residues can be divided into classes basedon common side chain properties:

-   -   hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;    -   neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   acidic: Asp, Glu;    -   basic: His, Lys, Arg;    -   residues that influence chain orientation: Gly, Pro; and    -   aromatic: Trp, Tyr, Phe.

Non-conservative substitutions can involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues can be introduced, for example, into regions of a humanantibody that are homologous with non-human antibodies, or into thenon-homologous regions of the molecule. Exemplary conservative aminoacid substitutions are set forth in Table B below.

TABLE B Original Preferred Residues Exemplary SubstitutionsSubstitutions Ala Va1, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln GlnAsp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala AlaHis Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine LeuLeu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyricacid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr LeuPro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp,Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu

As used herein, the terms “constant region” and “constant domain” areinterchangeable and have a meaning common in the art. The constantregion is an antibody portion, e.g., a carboxyl terminal portion of alight and/or heavy chain which is not directly involved in binding of anantibody to antigen but which can exhibit various effector functions,such as interaction with the Fc receptor. The constant region of animmunoglobulin molecule generally has a more conserved amino acidsequence relative to an immunoglobulin variable domain.

As used herein, the term “cross competes” means the situation in whichthe interaction between an antigen and a first antigen binding moleculeor binding fragment thereof blocks, limits, inhibits, or otherwisereduces the ability of a reference antigen binding molecule or bindingfragment thereof to interact with the antigen. Cross competition can becomplete, e.g., binding of the binding molecule to the antigencompletely blocks the ability of the reference binding molecule to bindthe antigen, or it can be partial, e.g., binding of the binding moleculeto the antigen reduces the ability of the reference binding molecule tobind the antigen. In certain embodiments, an antigen binding moleculethat cross competes with a reference antigen binding molecule binds thesame or an overlapping epitope as the reference antigen bindingmolecule. In other embodiments, the antigen binding molecule that crosscompetes with a reference antigen binding molecule binds a differentepitope than the reference antigen binding molecule. Numerous types ofcompetitive binding assays can be used to determine if one antigenbinding molecule competes with another, for example: solid phase director indirect radioimmunoassay (RIA); solid phase direct or indirectenzyme immunoassay (EIA); sandwich competition assay (Stahli et al.,(1983) Method Enzymol 9:242-53); solid phase direct biotin-avidin EIA(Kirkland et al., (1986) J Immunol 137:3614-19); solid phase directlabeled assay, solid phase direct labeled sandwich assay (Harlow andLane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press);solid phase direct label RIA using 1¹²⁵ label (Morel et al., (1988)Molec Immunol 25:7-15); solid phase direct biotin-avidin EIA (Cheung etal., (1990) Virology 176:546-52); and direct labeled RIA (Moldenhauer etal., (1990) Scand J Immunol 32:77-82).

The term “derivative” refers to a molecule that includes a chemicalmodification other than an insertion, deletion, or substitution of aminoacids (or nucleic acids). In certain embodiments, derivatives comprisecovalent modifications, including, but not limited to, chemical bondingwith polymers, lipids, or other organic or inorganic moieties. Incertain embodiments, a chemically modified antigen binding molecule (aderivative) can have a greater circulating half-life than an antigenbinding molecule that is not chemically modified. In some embodiments, aderivative antigen binding molecule is covalently modified to includeone or more water soluble polymer attachments, including, but notlimited to, polyethylene glycol, polyoxyethylene glycol, orpolypropylene glycol.

As used herein, the term “diabody” or dAB means bivalent antibodiescomprising two polypeptide chains, wherein each polypeptide chaincomprises VH and VL domains joined by a linker that is too short toallow for pairing between two domains on the same chain, thus allowingeach domain to pair with a complementary domain on another polypeptidechain (see, e.g., Holliger et al., (1993) Proc Natl Acad Sci U.S.A.90:6444-48, Poljak et al., (1994) Structure 2: 1121-23, and Perisic etal., (1994) Strucure 2(12): 1217-26). If the two polypeptide chains of adiabody are identical, then a diabody resulting from their pairing willhave two identical antigen binding sites. Polypeptide chains havingdifferent sequences can be used to make a diabody with two differentantigen binding sites. Similarly, tribodies and tetrabodies areantibodies comprising three and four polypeptide chains, respectively,and forming three and four antigen binding sites, respectively, whichcan be the same or different.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody can specificallybind. An epitope can be, for example, contiguous amino acids of apolypeptide (linear or contiguous epitope) or an epitope can, forexample, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope to whichan antibody binds can be determined by, e.g., NMR spectroscopy, X-raydiffraction crystallography studies, ELISA assays, hydrogen/deuteriumexchange coupled with mass spectrometry (e.g., liquid chromatographyelectrospray mass spectrometry), array-based oligo-peptide scanningassays, and/or mutagenesis mapping (e.g., site-directed mutagenesismapping). For X-ray crystallography, crystallization may be accomplishedusing any of the known methods in the art (e.g., Giege et al., (1994)Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson, (1990)Eur J Biochem 189: 1-23; Chayen, (1997) Structure 5: 1269-1274;McPherson, (1976) J Biol Chem 251: 6300-6303). Antibody:antigen crystalscan be studied using well known X-ray diffraction techniques and may berefined using computer software such as X-PLOR (Yale University, 1992,distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol(1985) Vols 114 & 115, eds Wyckoff et al.), and BUSTER (Bricogne, (1993)Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne, (1997)Meth Enzymol 276A: 361-423, ed. Carter; Roversi et al., (2000) ActaCrystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesismapping studies can be accomplished using any method known to one ofskill in the art. See, e.g., Champe et al., (1995) J Biol Chem 270:1388-94 and Cunningham & Wells, (1989) Science 244: 1081-85 for adescription of mutagenesis techniques, including alanine and argininescanning mutagenesis techniques.

As used herein, the term “Fab fragment” means is a monovalent fragmenthaving the VL, VH, CL and CH domains; a “F(ab′)₂ fragment” is a bivalentfragment having two Fab fragments linked by a disulfide bridge at thehinge region; a “Fv fragment” has the VH and VL domains of a single armof an antibody; and a “dAb fragment” has a VH domain, a VL domain, or anantigen-binding fragment of a VH or VL domain.

As used herein, the terms “immunospecifically binds,”“immunospecifically recognizes,” “specifically binds,” and “specificallyrecognizes” are analogous terms and are used interchangeably in thecontext of antigen binding molecules, and means that a given moleculepreferentially binds to an antigen (e.g., epitope or immune complex) assuch binding is understood by one skilled in the art. For example, anantigen binding molecule that specifically binds to an antigen may bindto other peptides or polypeptides, but with a comparatively loweraffinity as determined by, e.g., immunoassays, BIAcore®, KinExA 3000instrument (Sapidyne Instruments, Boise, Id.), or other assays known inthe art. In some embodiments, molecules that specifically bind to anantigen bind to the antigen with a K_(A) that is at least 2 logs, 2.5logs, 3 logs, 4 logs or greater than the K_(A) when the molecules bindto another antigen.

In another embodiment, molecules that specifically bind to an antigen(e.g., the anti-CD19 scFv FMC63; SEQ ID NO: 1), as well as moleculescomprising this sequence and cells presenting such molecules) bind witha dissociation constant (K_(d)) of about 1×10⁻⁷ M. In some embodiments,the antigen binding molecule specifically binds an antigen (e.g., theanti-CD19 scFv FMC63, as well as molecules comprising this sequence andcells presenting such molecules) with “high affinity” when the K_(d) isabout 1×10⁻⁹ M to about 5×10⁻⁹ M. In some embodiments, the antigenbinding molecule specifically binds an antigen (e.g., the anti-CD19 scFvFMC63, as well as molecules comprising this sequence and cellspresenting such molecules) with “very high affinity” when the K_(d) is1×10¹⁰ M to about 5×10⁻¹⁰ M.

In still another embodiment, molecules that specifically bind to anantigen (e.g., the anti-CD19 scFv FMC63, as well as molecules comprisingthis sequence and cells presenting such molecules) do not cross reactwith other proteins under similar binding conditions. In someembodiments, molecules that specifically bind to an antigen (e.g., theanti-CD19 scFv FMC63, as well as molecules comprising this sequence andcells presenting such molecules) do not cross react with other proteinsthat do not comprise the anti-CD19 scFv FMC63, molecules comprising thissequence and cells presenting such molecules. In some embodiments,provided herein is an antibody or fragment thereof that binds to theanti-CD19 scFv FMC63, as well as molecules comprising this sequence andcells presenting such molecules, with higher affinity than to anotherunrelated antigen. In certain embodiments, provided herein is an antigenbinding molecule (e.g., an antibody) or fragment thereof that binds tothe anti-CD19 scFv FMC63, as well as molecules comprising this sequenceand cells presenting such molecules, with a 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinitythan to another, unrelated antigen as measured by, e.g., aradioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.In some embodiments, the extent of binding of an antigen bindingmolecule, antibody or antigen binding fragment thereof that specificallybinds the anti-CD19 scFv FMC63, as well as molecules comprising thissequence and cells presenting such molecules, described herein comparedto an unrelated protein which does not comprise the anti-CD19 scFvFMC63, as well as molecules comprising this sequence and cellspresenting such molecules, is less than 10%, 15%, or 20% of the bindingof the antibody to linker fragment protein as measured by, e.g., aradioimmunoassay.

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃ and IgG₄.

As used herein, the term “immunoglobulin” means an immune molecule fromany of the commonly known isotypes, including but not limited to IgA,secretory IgA, IgG and IgM. IgG subclasses are also well known to thosein the art and include but are not limited to human IgG1, IgG2, IgG3 andIgG4. Many of the molecules described herein are immunoglobulins. Asused herein, “isotype” means the antibody class or subclass (e.g., IgMor IgG1) that is encoded by the heavy chain constant region genes.

An immunoglobulin is a tetrameric molecule, normally composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 130 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Berzofsky & Berkower, Ch. 7 in Fundamental Immunology (Paul, W., ed.,Lippincott Williams & Wilkins (2012); which chapter and volume isincorporated by reference in its entirety for all purposes). Thevariable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two primary bindingsites.

Naturally occurring immunoglobulin chains exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor “CDRs.” From N-terminus to C-terminus, both light and heavy chainscomprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain can be done in accordance withthe definitions of Kabat (see, e.g., Kabat et al. in Sequences ofProteins of Immunological Interest, 5th Ed., NIH Publication 91-3242,Bethesda Md. (1991)) or Chothia (Chothia, used herein, (see, e.g.,Chothia & Lesk (1987), J. Mol. Biol. 196:901-917; Chothia et al., 1989,Nature 342:878-883 or Honegger & Pluckthun (2001), J Mol Biol309:657-670). The Kabat, Chothia, IGMT and Abm (Oxford Molecular)numbering systems are described more fully herein.

As used herein, the term “in vitro cell” refers to any cell that iscultured ex vivo. An in vitro cell can include a human cell such as a Tcell or dendritic cell, or it can include CHO, sP2/0, rabbit and othernon-human cells.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are known in the art. In specific embodiments, thelight chain is a human light chain.

The term “neutralizing” refers to an antigen binding molecule, scFv,antibody, or a fragment thereof, that binds to a ligand (e.g., a moietycomprising the anti-CD19 scFv FMC63, as well as molecules comprisingthis sequence and cells presenting such molecules) and prevents orreduces the biological effect of that ligand. In some embodiments, theantigen binding molecule, scFv, antibody, or a fragment thereof,directly blocking a binding site on the ligand or otherwise alters theligand's ability to bind through indirect means (such as structural orenergetic alterations in the ligand). In some embodiments, the antigenbinding molecule, scFv, antibody, or a fragment thereof prevents theprotein to which it is bound from performing a biological function.

As used herein, the term “patient” means any human who is being treatedfor an abnormal physiological condition, such as cancer or has beenformally diagnosed with a disorder, those without formally recognizeddisorders, those receiving medical attention, those at risk ofdeveloping the disorders, etc. The terms “subject” and “patient” areused interchangeably herein and include both human and non-human animalsubjects.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably herein, and mean a compound comprising amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, but no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. The term polypeptide encompasses any peptide or proteincomprising two or more amino acids joined to each other by peptidebonds. As used herein, the term refers to both short chains, which alsocommonly are referred to as peptides, oligopeptides and oligomers, andto longer chains, which generally are referred to as proteins.“Polypeptides” include, for example, biologically active fragments,substantially homologous polypeptides, oligopeptides, homodimers,heterodimers, variants of polypeptides, modified polypeptides,derivatives, analogs, fusion proteins, among others. The term“polypeptide” includes natural peptides, recombinant peptides, syntheticpeptides, or a combination thereof.

In some aspects, the polypeptides and/or proteins have deletions from,additions to, and/or substitutions of one or more amino acids of antigenbinding molecule. Useful polypeptide fragments may includeimmunologically functional fragments of antigen binding molecules,including not limited to one or more CDR regions, variable domains of aheavy and/or light chain, a portion of other portions of an antibodychain, and the like. Moieties that can be substituted for one or moreamino acids of an antigen binding molecule include, e.g., D or L formsof amino acids, an amino acid different from the amino acid normallyfound in the same position of an antigen binding molecule (relative toSEQ ID NOs: 2-73), deletions, non-naturally occurring amino acids, andchemical analogs of amino acids.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide and form an aspect of the instant disclosure. These types ofnon-peptide compound are termed “peptide mimetics” or “peptidomimetics.”See, e.g., Fauchere, (1986) Adv. Drug Res. (Testa, ed.) 15:29-69; Veber& Freidinger, (1985) TINS, p. 392; and Evans et al., (1987) J. Med.Chem, 30:1229-39, which are incorporated herein by reference for anypurpose.

Polypeptides, peptides, proteins and analogous molecules comprising theanti-CD19 scFv FMC63, as well as molecules comprising this sequence andcells presenting such molecules, are specifically encompassed by theterms.

As used herein, the term “percent identity” means the percent ofidentical residues between the amino acids or nucleotides in thecompared molecules. For these calculations, gaps in alignments (if any)must be addressed by a particular mathematical model or computer program(i.e., an “algorithm”). Methods that can be used to calculate theidentity of the aligned nucleic acids or polypeptides include thosedescribed in Computational Molecular Biology, (Lesk, ed.), (1988) NewYork: Oxford University Press; Biocomputing Informatics and GenomeProjects, (Smith, ed.), 1993, New York: Academic Press; ComputerAnalysis of Sequence Data, Part I, (Griffin and Griffin, eds.), 1994,New Jersey: Humana Press; von Heinje, (1987) Sequence Analysis inMolecular Biology, New York: Academic Press; Sequence Analysis Primer,(Gribskov and Devereux, eds.), 1991, New York: M. Stockton Press; andCarillo et al., (1988) J. Applied Math. 48:1073.

In calculating percent identity, the sequences being compared arealigned in a way that gives the largest match between the sequences. Thecomputer program used to determine percent identity can be, e.g., MOE(Chemical Computing Group) or DNASTAR (University of Wisconsin, Madison,Wis.). The computer algorithm GAP can be used to align the twopolypeptides or polynucleotides for which the percent sequence identityis to be determined. The sequences are aligned for optimal matching oftheir respective amino acid or nucleotide (the “matched span,” asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal, wherein the “average diagonal” is theaverage of the diagonal of the comparison matrix being used; the“diagonal” is the score or number assigned to each perfect amino acidmatch by the particular comparison matrix) and a gap extension penalty(which is usually 1/10 times the gap opening penalty), as well as acomparison matrix such as PAM 250 or BLOSUM 62 are used in conjunctionwith the algorithm. In certain embodiments, a standard comparison matrix(see, e.g., Dayhoff et al., (1978) Atlas of Protein Sequence andStructure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al.,(1992) Proc. Natl. Acad. Sci. U.S.A. 89: 10915-10919 for the BLOSUM 62comparison matrix) is also used by the algorithm.

Certain alignment schemes for aligning two amino acid sequences canresult in matching of only a short region of the two sequences, and thissmall aligned region can have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (e.g., the GAPprogram) can be adjusted if desired to result in an alignment that spansat least 50 contiguous amino acids of the target polypeptide.

As used herein, the terms “single-chain antibody” and “single chainfragment variable (scFv)” are used interchangeably and mean an antigenbinding molecule in which a VL and a VH region are joined via a linkerto form a continuous protein chain wherein the linker is long enough toallow the protein chain to fold back on itself and form a monovalentantigen binding site (see, e.g., Bird et al., (1988) Science 242:423-26and Huston et al., (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83(1988). FMC63 is a specific example of a scFv.

A “therapeutically effective amount,” “effective dose,” “effectiveamount,” or “therapeutically effective dosage” of a therapeutic agent,(e.g., a moiety comprising the anti-CD19 scFv FMC63, as well asmolecules comprising this sequence and cells presenting such molecules),is any amount that, when used alone or in combination with anothertherapeutic agent, protects a subject against the onset of a disease orpromotes disease regression evidenced by a decrease in severity ofdisease symptoms, an increase in frequency and duration of diseasesymptom-free periods, or a prevention of impairment or disability due tothe disease affliction. The ability of a therapeutic agent to promotedisease regression can be evaluated using a variety of methods known tothe skilled practitioner, such as in human subjects during clinicaltrials, in animal model systems predictive of efficacy in humans, or byassaying the activity of the agent in in vitro assays.

The terms “transduction” and “transduced” refer to the process wherebyforeign DNA is introduced into a cell via viral vector (see Hard andJones (1997) “Genetics: Principles and Analysis,” 4^(th) ed, Jones &Bartlett). In some embodiments, the vector is a retroviral vector, a DNAvector, a RNA vector, an adenoviral vector, a baculoviral vector, anEpstein Barr viral vector, a papovaviral vector, a vaccinia viralvector, a herpes simplex viral vector, an adenovirus associated vector,a lentiviral vector, or any combination thereof.

As used herein, the terms “variable region” or “variable domain” areused interchangeably and mean a portion of an antibody, generally, aportion of a light or heavy chain, typically about the amino-terminalend of the antibody and comprising about 100-130 amino acids in theheavy chain and about 90 to 115 amino acids in the light chain, whichdiffer extensively in sequence among antibodies and are used in thebinding and specificity of a particular antibody for its particularantigen. The variability in sequence is concentrated in those regionscalled complementarity determining regions (CDRs) while the more highlyconserved regions in the variable domain are called framework regions(FR). The CDRs of the light and heavy chains are primarily responsiblefor the interaction and specificity of the antibody with antigen.

In certain embodiments, the variable region of an antigen bindingmolecule is a human variable region. In further embodiments, thevariable region comprises rodent, human or murine CDRs and humanframework regions (FRs). In further embodiments, the variable region isa primate (e.g., a non-human primate) variable region. In yet furtherembodiments, the variable region is a rabbit variable region. In otherembodiments, the variable region comprises human CDRs and non-human(e.g., rabbit, murine, rat or non-human primate) framework regions(FRs). In other embodiments, the variable region comprises non-human(e.g., rabbit, murine, rat or non-human primate) CDRs and humanframework regions (FRs).

The terms “VH,” “VH domain” and “VH chain” are used interchangeably andmean the heavy chain variable region of an antigen binding molecule,antibody or an antigen binding fragment thereof.

The terms “VL,” “VL domain” and “VL chain” are used interchangeably andmean the light chain variable region of an antigen binding molecule,antibody or an antigen binding fragment thereof.

Various aspects of the invention are described in further detail in thefollowing subsections.

II. Antigen Binding Molecules and Polynucleotides Encoding the Same

The present disclosure is directed to antigen binding molecules,including antibodies, that specifically bind the anti-CD19 scFv FMC63(SEQ ID NO: 1), as well as molecules comprising this sequence and cellspresenting such molecules, and/or those which cross compete with one ormore antigen binding molecules described herein (i.e., one or more ofthose described in FIGS. 5-21 and/or disclosed in the appended SequenceListing). Polynucleotides encoding the antigen binding molecules arealso provided, and form an aspect of the instant disclosure.

An antibody or antigen binding molecule encoded of the present inventioncan be single chained or double chained. In some embodiments, theantibody or antigen binding molecule is single chained. In certainembodiments, the antigen binding molecule is selected from the groupconsisting of an scFv, a Fab, a Fab′, a Fv, a F(ab′)₂, a dAb, and anycombination thereof. In one particular embodiment, the antibody orantigen binding molecule comprises an scFv.

In certain embodiments, an antigen binding molecule such as an antibodycomprises a single chain, wherein the heavy chain variable region andthe light chain variable region are connected by a linker (e.g., anscFv). In some embodiments, the VH is located at the N terminus of thelinker and the VL is located at the C terminus of the linker. In otherembodiments, the VL is located at the N terminus of the linker and theVH is located at the C terminus of the linker. In some embodiments, thelinker comprises at least about 5, at least about 8, at least about 10,at least about 13, at least about 15, at least about 18, at least about20, at least about 25, at least about 30, at least about 35, at leastabout 40, at least about 45, at least about 50, at least about 60, atleast about 70, at least about 80, at least about 90, or at least about100 amino acids. In some embodiments, the linker comprises between about8 amino acids and about 18 amino acids (e.g., 10 amino acids).

In some embodiments, the antigen binding molecules of the presentinvention specifically bind to the anti-CD19 scFv FMC63, as well asmolecules comprising this sequence and cells presenting such molecules.In certain embodiments, an antigen binding molecule of the presentdisclosure specifically binds the anti-CD19 scFv FMC63, as well asmolecules comprising this sequence and cells presenting such moleculeswith a K_(D) of less than 1×10⁻⁶ M, less than 1×10⁻⁷ M, less than 1×10⁻⁸M, or less than 1×10⁻⁹ M. In one particular embodiment, an antigenbinding molecule specifically binds to the anti-CD19 scFv FMC63, as wellas molecules comprising this sequence and cells presenting suchmolecules, with a K_(D) of less than 1×10⁻⁷ M. In another embodiment, anantigen binding molecule specifically binds the anti-CD19 scFv FMC63, aswell as molecules comprising this sequence and cells presenting suchmolecules, with a K_(D) of less than 1×10⁻⁸ M. In some embodiments, anantigen binding molecule binds the anti-CD19 scFv FMC63, as well asmolecules comprising this sequence and cells presenting such molecules,with a K_(D) of about 1×10⁻⁷ M, about 2×10⁻⁷M, about 3×10⁻⁷M, about4×10⁻⁷M, about 5×10⁻⁷M, about 6×10⁻⁷M, about 7×10⁻⁷ M, about 8×10⁻⁷ M,about 9×10⁻⁷ M, about 1×10⁻⁸ M, about 2×10⁻⁸ M, about 3×10⁻⁸ M, about4×10⁻⁸ M, about 5×10⁻⁸ M, about 6×10⁻⁸ M, about 7×10⁻⁸ M, about 8×10⁻⁸M, about 9×10⁻⁸ M, about 1×10⁻⁹ M, about 2×10⁻⁹ M, about 3×10⁻⁹ M, about4×10⁻⁹ M, about 5×10⁻⁹M, about 6×10⁻⁹M, about 7×10⁻⁹M, about 8×10⁻⁹M,about 9×10⁻⁹M, about 1×10⁻¹⁰ M, or about 5×10⁻¹⁰ M. K_(D) can becalculated using standard methodologies, as described herein.

In specific embodiments, an antigen binding molecule of the instantdisclosure is an antibody identified herein as Clone 7, Clone 13, Clone14-1, Clone 14-7, Clone 15, or Clone 17, and each comprises thefollowing heavy and light chain amino acid, coding, variable, and CDRsequences, as provided and labeled:

Clone 7 VH DNA (SEQ ID NO: 2)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGGAG CAGCTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGAGGAACCCTGACAGTCACCTGCAAAGCCTCTGGATTCTCCTTCAGTAACAATGGAATTTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGTCTTTATGTTGGTAGTAGTGATACCACTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAAGCTCGTCGACCACGGTGACTCTACAAATGACCAGTCTGACAGTCGCGGACACGGCCACCTATTTCTGTACGATAAATCTCGGCTTGTGGGGCCCCGGCACCCTGGTCACCGTCTCCTCAClone 7 VH AA (CDRs underlined) (SEQ ID NO: 3)METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPGGTLTVTCKASGFSFSNNGICWVRQAPGKGLEWIGCLYVGSSDTTYYASWAKGRFTISKSSSTTVTLQMTSLTVADTATYFCTINLGLWGPGTLVTVSSClone 7 HC AA (CDRs underlined) (SEQ ID NO: 4)METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPGGTLTVTCKASGFSFSNNGICWVRQAPGKGLEWIGCLYVGSSDTTYYASWAKGRFTISKSSSTTVTLQMTSLTVADTATYFCTINLGLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKClone 7 VH CDR1 AA (SEQ ID NO: 5) GFSFSNN Clone 7 VH CDR2 AA(SEQ ID NO: 6) YVGSSD Clone 7 VH CDR3 AA (SEQ ID NO: 7) NLGLClone 7VL DNA (SEQ ID NO: 8)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCATCGTGGTGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGAGTGTTTATAATAGCGACTGGTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATGCTGCATCCACTCTGGCATCTGGGGTCCCATCGCGCTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGATGTGGTGTGTGACGATGCTGCCACTTATTATTGTGCAGGATATAAAAGTAGTAGTACTGATGGGATTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA Clone 7 VL AA (CDRs underlined) (SEQ ID NO: 9)MDTRAPTQLLGLLLLWLPGATFAIVVTQTPSSKSVPVGGTVTINCQASESVYNSDWLAWYQQKPGQPPKQLIYAASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSSSTDGIAFGGGTEVVVKClone 7 LC AA (CDRs underlined) (SEQ ID NO: 10)MDTRAPTQLLGLLLLWLPGATFAIVVTQTPSSKSVPVGGTVTINCQASESVYNSDWLAWYQQKPGQPPKQLIYAASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSSSTDGIAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDCClone 7 VL CDR1 AA (SEQ ID NO: 11) QASESVYNSDWLA Clone 7 VL CDR2 AA(SEQ ID NO: 12) AASTLAS Clone 7 VL CDR3 AA (SEQ ID NO: 13) AGYKSSSTDGIAClone 13 VH DNA (SEQ ID NO: 14)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGGAGCAGCTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGAGGAACCCTGACAGTCACCTGCAAAGCCTCTGGATTCTCCTTCAGTAACAATGGAATTTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGTCTTTATGTTGGTAGTAGTGATACCACTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAAGCTCGTCGACCACGGTGACTCTACAAATGACCAGTCTGACAGTCGCGGACACGGCCACCTATTTCTGTACGATAAATCTCGGCTTGTGGGGCCCCGGCACCCTGGTCACCGTCTCCTCAClone 13 VH AA (CDRs underlined) (SEQ ID NO: 15)METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPGGTLTVTCKASGFSFSNNGICWVRQAPGKGLEWIGCLYVGSSDTTYYASWAKGRFTISKSSSTTVTLQMTSLTVADTATYFCTINLGLWGPGTLVTVSSClone 13 HC AA (CDRs underlined) (SEQ ID NO: 16)METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPGGTLTVTCKASGFSFSNNGICWVRQAPGKGLEWIGCLYVGSSDTTYYASWAKGRFTISKSSSTTVTLQMTSLTVADTATYFCTINLGLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKClone 13 VH CDR1 AA (SEQ ID NO: 5) GFSFSNN Clone 13 VH CDR2 AA(SEQ ID NO: 6) YVGSSD Clone 13 VH CDR3 AA (SEQ ID NO: 7) NLGLClone 13 VL DNA (SEQ ID NO: 17)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACACTTGCCATCGTGGTGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGAGTGTTTATAATAGCGACTGGTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATGCTGCATCCACTCTGGCATCTGGGGTCCCATCGCGCTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGATGTGGTGTGTGACGATGCTGCCACTTATTATTGTGCAGGATATAAAAGTAGTAGTACTGATGGGATTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA Clone 13 VL AA (CDRs underlined) (SEQ ID NO: 18)MDTRAPTQLLGLLLLWLPGATLAIVVTQTPSSKSVPVGGTVTINCQASESVYNSDWLAWYQQKPGQPPKQLIYAASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSSSTDGIAFGGGTEVVVKClone 13 LC AA (CDRs underlined) (SEQ ID NO: 19)MDTRAPTQLLGLLLLWLPGATLAIVVTQTPSSKSVPVGGTVTINCQASESVYNSDWLAWYQQKPGQPPKQLIYAASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSSSTDGIAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDCClone 13 VL CDR1 AA (SEQ ID NO: 11) QASESVYNSDWLA Clone 13 VL CDR2 AA(SEQ ID NO: 12) AASTLAS Clone 13 VL CDR3 AA (SEQ ID NO: 13) AGYKSSSTDGIAClone 14-1 VH DNA (SEQ ID NO: 20)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGGAGCAGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCAAAGCCTCTGGATTCGACTTCAGTATCAACTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGTTGGAGTGGATCGCATGCATTTATACTGGTGATGATGACACTTTCTACGCGAGCTGGGCGAAAGGCCGGTTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTACAACTGAACAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGTGAGAGGTCTATATAGTGGTAGTATTAATAACCTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA Clone 14-1 VH AA (CDRs underlined) (SEQ ID NO: 21)METGLRWLLLVAVLKGVQCQEQLEESGGGLVKPGASLTLTCKASGFDFSINYYMCWVRQAPGKGLEWIACIYTGDDDTFYASWAKGRFTISKTSSTTVTLQLNSLTAADTATYFCVRGLYSGSINNLWGPGTLVTVSSClone 14-1 HC AA (CDRs underlined) (SEQ ID NO: 22)METGLRWLLLVAVLKGVQCQEQLEESGGGLVKPGASLTLTCKASGFDFSINYYMCWVRQAPGKGLEWIACIYTGDDDTFYASWAKGRFTISKTSSTTVTLQLNSLTAADTATYFCVRGLYSGSINNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKClone 14-1 VH CDR1 AA (SEQ ID NO: 23) GPDFSINY Clone 14-1 VH CDR2 AA(SEQ ID NO: 24) YTGDD Clone 14-1 VH CDR3 AA (SEQ ID NO: 25) GLYSGSINNLClone 14-1VL DNA (SEQ ID NO: 26)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGATGCCAGATGTGCGCTTGTGATGACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGGCCAGTCAGAGTGTTTATAACAACGACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAACTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCGCTTACTATTGTGCAGGCGTTAAAGGTTATAGTAATGATAATAATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA Clone 14-1 VL AA (CDRs underlined) (SEQ ID NO: 27)MDTRAPTQLLGLLLLWLPDARCALVMTQTPSPVSAAVGGTVTISCQASQSVYNNDYLSWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAAAYYCAGVKGYSNDNNGFGGGTEVVVKClone 14-1 LC AA (CDRs underlined) (SEQ ID NO: 28)MDTRAPTQLLGLLLLWLPDARCALVMTQTPSPVSAAVGGTVTISCQASQSVYNNDYLSWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAAAYYCAGVKGYSNDNNGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDCClone 14-1 VL CDR1 AA (SEQ ID NO: 29) QASQSVYNNDYLSClone 14-1 VL CDR2 AA (SEQ ID NO: 30) YASTLAS Clone 14-1 VL CDR3 AA(SEQ ID NO: 31) AGVKGYSNDNNG Clone 14-7 VH DNA (SEQ ID NO: 32)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAATGTCAGTCGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCAAAGCCTCTGGATTCGACTTCAGTATCAACTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGTTGGAGTGGATCGCATGCATTTATACTGGTGATGATGACACTTTCTACGCGAGCTGGGCGAAAGGCCGGTTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTACAACTGAACAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGTGAGAGGTCTATATAGTGGTAGTATTAATAACCTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA Clone 14-7 VH AA (CDRs underlined) (SEQ ID NO: 33)METGLRWLLLVAVLKGVQCQSLEESGGGLVKPGASLTLTCKASGFDFSINYYMCWVRQAPGKGLEWIACIYTGDDDTFYASWAKGRFTISKTSSTTVTLQLNSLTAADTATYFCVRGLYSGSINNLWGPGTLVTVSSClone 14-7 HC AA (CDRs underlined) (SEQ ID NO: 34)METGLRWLLLVAVLKGVQCQSLEESGGGLVKPGASLTLTCKASGFDFSINYYMCWVRQAPGKGLEWIACIYTGDDDTFYASWAKGRFTISKTSSTTVTLQLNSLTAADTATYFCVRGLYSGSINNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKClone 14-7 VH CDR1 AA (SEQ ID NO: 23) GPDFSINY Clone 14-7 VH CDR2 AA(SEQ ID NO: 24) YTGDD Clone 14-7 VH CDR3 AA (SEQ ID NO: 25) GLYSGSINNLClone 14-7VL DNA (SEQ ID NO: 35)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGATGCCAGATGTGCGCTTGTGATGACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGGCCAGTCAGAGTGTTTATAACAACGACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAACTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCGCTTACTATTGTGCAGGCGTTAAAGGTTATAGTAATGATAATAATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA Clone 14-7 VL AA (CDRs underlined) (SEQ ID NO: 36)MDTRAPTQLLGLLLLWLPDARCALVMTQTPSPVSAAVGGTVTISCQASQSVYNNDYLSWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAAAYYCAGVKGYSNDNNGFGGGTEVVVKClone 14-7 LC AA (CDRs underlined) (SEQ ID NO: 37)MDTRAPTQLLGLLLLWLPDARCALVMTQTPSPVSAAVGGTVTISCQASQSVYNNDYLSWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAAAYYCAGVKGYSNDNNGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDCClone 14-7 VL CDR1 AA (SEQ ID NO: 29) QASQSVYNNDYLSClone 14-7 VL CDR2 AA (SEQ ID NO: 30) YASTLAS Clone 14-7 VL CDR3 AA(SEQ ID NO: 31) AGVKGYSNDNNG Clone 15 VH DNA (SEQ ID NO: 38)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGGGTCCAGTGTCAGTCGTTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCTTCACGAGCAACTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCGTGCATTTTTCTTGGTAGTAGTGGTAACACTGTCTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGTCGCGGACACGGCCACCTATTTCTGTGCGAGAGACTATGTTAATGGTTATGACTACTTTAACTTGTGGGGCCCAGGCACCTTGGTCACCGTCTCCTCA Clone 15 VH AA (CDRs underlined) (SEQ ID NO: 39)METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCTASGFSFTSNYYMCWVRQAPGKGLEWVACIFLGSSGNTVYANWAKGRFTISKTSSTTVTLQMTSLTVADTATYFCARDYVNGYDYFNLWGPGTLVTVSSClone 15 HC AA (CDRs underlined) (SEQ ID NO: 40)METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCTASGFSFTSNYYMCWVRQAPGKGLEWVACIFLGSSGNTVYANWAKGRFTISKTSSTTVTLQMTSLTVADTATYFCARDYVNGYDYFNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKClone 15 VH CDR1 AA (SEQ ID NO: 41) GFSFTSNY Clone 15 VH CDR2 AA(SEQ ID NO: 42) FLGSSG Clone 15 VH CDR3 AA (SEQ ID NO: 43) DYVNGYDYFNLClone 15 VL DNA (SEQ ID NO: 44)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCGGCTGTTGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATAATAAGAACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAAGGCCTGATCTATTCTACATCGACTCTAGATTCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCAGTTATGATTGTAGTAGTGCTGATTGTAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA Clone 15 VL AA (CDRs underlined) (SEQ ID NO: 45)MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAAVGGTVTINCQSSQSVYNKNLAWYQQKPGQPPKGLIYSTSTLDSGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSSADCNAFGGGTEVVVKClone 15 LC AA (CDRs underlined) (SEQ ID NO: 46)MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAAVGGTVTINCQSSQSVYNKNLAWYQQKPGQPPKGLIYSTSTLDSGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSSADCNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDCClone 15 VL CDR1 AA (SEQ ID NO: 47) QSSQSVYNKNLA Clone 15 VL CDR2 AA(SEQ ID NO: 48) STSTLDS Clone 15 VL CDR3 AA (SEQ ID NO: 49)LGSYDCSSADCNA Clone 17 VH DNA (SEQ ID NO: 50)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAATGTCAGTCGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCTTCAGTGACAGTTGGTACTTGTGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTTATACTGGTGATGGTGACACTTATTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAGACCTCGTCGACCACAGTGACTCTACAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGGGGTGCCCAATTTTACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAClone 17 VH AA (CDRs underlined) (SEQ ID NO: 51)METGLRWLLLVAVLKGVQCQSLEESGGGLVKPGASLTLTCTASGFSFSDSWYLCWVRQAPGKGLEWIACIYTGDGDTYYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGAQFYLWGQGTLVTVSSClone 17 HC AA (CDRs underlined) (SEQ ID NO: 52)METGLRWLLLVAVLKGVQCQSLEESGGGLVKPGASLTLTCTASGFSFSDSWYLCWVRQAPGKGLEWIACIYTGDGDTYYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGAQFYLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKClone 17 VH CDR1 AA (SEQ ID NO: 53) GFSFSDSW Clone 17 VH CDR2 AA(SEQ ID NO: 54) YTGDG Clone 17 VH CDR3 AA (SEQ ID NO: 55) GAQFYLClone 17 VL DNA (SEQ ID NO: 56)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAGGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATGCCAACACCTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTGCATCCAGTCTGGCATCTGGGGTCCCACCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCGCTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCAGATATAGTTGTGGTCTTGCTGATTGTGCTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA Clone 17 VL AA (CDRs underlined) (SEQ ID NO: 57)MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAAVGGTVTINCQSSQSVYANTYLSWYQQKPGQPPKQLIYSASSLASGVPPRFKGSGSGTQFALTISDVQCDDAATYYCLGRYSCGLADCAAFGGGTEVVVKClone 17 LC AA (CDRs underlined) (SEQ ID NO: 58)MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAAVGGTVTINCQSSQSVYANTYLSWYQQKPGQPPKQLIYSASSLASGVPPRFKGSGSGTQFALTISDVQCDDAATYYCLGRYSCGLADCAAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDCClone 17 VL CDR1 AA (SEQ ID NO: 59) QSSQSVYANTYLS Clone 17 VL CDR2 AA(SEQ ID NO: 60) SASSLAS Clone 17 VL CDR3 AA (SEQ ID NO: 61)LGRYSCGLADCAA

In some embodiments, the antigen binding molecules of the presentdisclosure are antibodies and antigen binding fragments thereof. In someembodiments, the antibodies of the present disclosure comprise at leastone CDR set forth in FIGS. 5-21. In another aspect, the presentdisclosure provides hybridomas capable of producing the antibodiesdisclosed herein, and also methods of producing antibodies fromhybridomas, as described herein and as known in the art.

Humanized antibodies are described herein and may be prepared by knowntechniques. In some embodiments, a humanized monoclonal antibodycomprises the variable domain of a murine or rabbit antibody (or all orpart of the antigen binding site thereof) and a constant domain derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise an antigen binding site of a murine or rabbit monoclonalantibody and a variable domain fragment (lacking the antigen bindingsite) derived from a human antibody. Procedures for the production ofengineered monoclonal antibodies include those described in Riechmann etal., (1988) Nature 332:323, Liu et al., (1987) Proc. Nat. Acad. Sci. USA84:3439, Larrick et al., (1989) Bio/Technology 7:934, and Winter et al.,(1993) TIPS 14:139. In some embodiments, the chimeric antibody is a CDRgrafted antibody. Techniques for humanizing antibodies are discussed in,e.g., U.S. Pat. Nos. 5,869,619; 5,225,539; 5,821,337; 5,859,205;6,881,557; Padlan et al., (1995) FASEB J. 9:133-39; Tamura et al.,(2000) J. Immunol. 164:1432-41; Zhang et al., (2005) Mol. Immunol.42(12):1445-1451; Hwang et al., Methods. (2005) 36(1):35-42; Dall'Acquaet al., (2005) Methods 36(1):43-60; and Clark, (2000) Immunology Today21(8):397-402.

An antigen binding molecule of the present invention can also be a fullyhuman monoclonal antibody. Fully human monoclonal antibodies can begenerated by any number of techniques with which those having ordinaryskill in the art will be familiar Such methods include, but are notlimited to, Epstein Barr Virus (EBV) transformation of human peripheralblood cells (e.g., containing B lymphocytes), in vitro immunization ofhuman B-cells, fusion of spleen cells from immunized transgenic micecarrying inserted human immunoglobulin genes, isolation from humanimmunoglobulin V region phage libraries, or other procedures as known inthe art and based on the disclosure herein.

Procedures have been developed for generating human monoclonalantibodies in non-human animals. For example, mice in which one or moreendogenous immunoglobulin genes have been inactivated by various meanshave been prepared. Human immunoglobulin genes have been introduced intothe mice to replace the inactivated mouse genes. In this technique,elements of the human heavy and light chain locus are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci(see also Bruggemann et al., (1997) Curr. Opin. Biotechnol. 8:455-58).

Examples of techniques for production and use of transgenic animals forthe production of human or partially human antibodies are described inU.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806; Davis et al.,Antibody Engineering: Methods and Protocols, (Lo, ed) Humana Press, NJ,191-200 (2003); Kellermann et al., (2002) Curr Opin Biotechnol.13:593-97; Russel et al., (2000) Infect Immun. 68:1820-26; Gallo et al.,(2000) Eur J. Immun. 30:534-40; Davis et al., (1999) Cancer MetastasisRev. 18:421-25; Green, (1999) J Immunol Methods 231:11-23; Jakobovits,(1998) Advanced Drug Delivery Reviews 31:33-42; Green et al., (1998) JExp Med. 188:483-95; Jakobovits, (1998) Exp. Opin. Invest. Drugs.7:607-14; Tsuda et al., (1997) Genomics, 42:413-21; Mendez et al.,(1997) Nat. Genet. 15:146-56; Jakobovits, (1994) Curr Biol. 4:761-63;Arbones et al., (1994) Immunity 1:247-60; Green et al., (1994) Nat.Genet. 7:13-21; Jakobovits et al., (1993) Nature 362:255-58; Jakobovitset al., (1993) Proc Natl Acad Sci USA 90:2551-55; Chen et al., (1993)Intl Immunol 5:647-656; Choi et al., (1993) Nature Genetics 4:117-23;Fishwild et al., (1996) Nature Biotechnology 14:845-51; Lonberg et al.,(1994) Nature 368: 856-59; Lonberg, (1994) Handbook of ExperimentalPharmacology 113: 49-101; Neuberger, (1996) Nature Biotech 14:826;Taylor et al., (1992) Nucleic Acids Research 20:6287-95; Taylor et al.,(1994) Intl Immunol 6:579-91; Tomizuka et al., (1997) Nature Genetics16:133-43; Tomizuka et al., (2000) Proc Nat Acad Sci USA 97:722-27;Tuaillon et al., (1993) Proc Nat Acad Sci USA 90:3720-24; Tuaillon etal., (1994) J Immunol 152:2912-20; Lonberg et al., (1994) Nature368:856; Taylor et al., (1994) Intl Immunol 6:579; U.S. Pat. No.5,877,397; Bruggemann et al., (1997) Curr. Opin. Biotechnol. 8:455-58;Jakobovits et al., (1995) Ann. N.Y. Acad. Sci. 764:525-35.

An additional method for obtaining antigen binding molecules of theinvention is by the use of phage display, which is well-established forthis purpose. See, e.g., Winter et al., (1994) Ann. Rev. Immunol.12:433-55; Burton et al., (1994) Adv. Immunol 57:191-280. Human ormurine immunoglobulin variable region gene combinatorial libraries canbe created in phage vectors that can be screened to select Ig fragments(Fab, Fv, sFv, or multimers thereof) that bind the anti-CD19 scFv FMC63,as well as molecules comprising this sequence and cells presenting suchmolecules. See, e.g., U.S. Pat. No. 5,223,409; Huse et al., (1989)Science 246:1275-81; Sastry et al., (1989) Proc. Natl. Acad. Sci. USA86:5728-32; Alting-Mees et al., (1990) Strategies in Molecular Biology3:1-9; Kang et al., (1991) Proc. Natl. Acad. Sci. USA 88:4363-66;Hoogenboom et al., (1992) J. Mol. Biol. 227:381-388; Schlebusch et al.,(1997) Hybridoma 16:47-52 and references cited therein. For example, alibrary containing a plurality of polynucleotide sequences encoding Igvariable region fragments can be inserted into the genome of afilamentous bacteriophage, such as M13 or lambda phage (λImmunoZap™(H)and λImmunoZap™(L) vectors (Stratagene, La Jolla, Calif.) can also beused in this approach) or a variant thereof, in frame with the sequenceencoding a phage coat protein.

Briefly, mRNA is isolated from a B-cell population, and used to createheavy and light chain immunoglobulin cDNA expression libraries in theλImmunoZap™(H) and λImmunoZap™(L) vectors. These vectors can be screenedindividually or co-expressed to form Fab fragments or antibodies.Positive plaques can subsequently be converted to a non-lytic plasmidthat allows high level expression of monoclonal antibody fragments fromE. coli.

In some embodiments, in a hybridoma the variable regions of a geneexpressing a monoclonal antibody of interest are amplified usingnucleotide primers. These primers can be synthesized by one of ordinaryskill in the art, or can be purchased from commercial sources, whichalso sell primers for mouse and human variable regions including, amongothers, primers for V_(Ha), V_(Hb), V_(Hc), V_(Hd), C_(Hl), V_(L) andC_(L) regions). These primers can be used to amplify heavy or lightchain variable regions, which can then be inserted into vectors such asλImmunoZap™(H) and λImmunoZap™(L) (Stratagene), respectively. Thesevectors can then be introduced into E. coli, yeast, or mammalian-basedsystems for expression. Large amounts of a single-chain proteincontaining a fusion of the V_(H) and V_(L) domains can be produced usingthese methods.

Once cells producing the antigen binding molecules provided herein havebeen obtained using any of the above-described immunization and othertechniques, the specific antibody genes can be cloned by isolating andamplifying DNA or mRNA therefrom according to standard procedures asdescribed herein. The antibodies produced therefrom can be sequenced andthe CDRs identified and the DNA coding for the CDRs can be manipulatedas described previously to generate other antibodies according to theinvention.

It will be understood by one skilled in the art that some proteins, suchas antibodies, can undergo a variety of posttranslational modifications.The type and extent of these modifications often depends on the hostcell line used to express the protein as well as the culture conditions.Such modifications can include variations in glycosylation, methionineoxidation, diketopiperizine formation, aspartate isomerization andasparagine deamidation. A frequent modification is the loss of acarboxy-terminal basic residue (such as lysine or arginine) due to theaction of carboxypeptidases (as described in Harris, (1995) J Chromatog705:129-34.

An alternative method for production of a murine monoclonal antibody(from which FMC63 can be derived) is to inject the hybridoma cells intothe peritoneal cavity of a syngeneic mouse, for example, a mouse thathas been treated (e.g., pristane-primed) to promote formation of ascitesfluid containing the monoclonal antibody. Monoclonal antibodies can beisolated and purified by a variety of well-established techniques. Suchisolation techniques include affinity chromatography with Protein-ASepharose, size-exclusion chromatography, and ion-exchangechromatography (see, e.g., Baines and Thorpe, (1992) in Methods inMolecular Biology, 10:79-104 (The Humana Press). Monoclonal antibodiescan be purified by affinity chromatography using an appropriate ligandselected based on particular properties of the antibody (e.g., heavy orlight chain isotype, binding specificity, etc.). Examples of a suitableligand, immobilized on a solid support, include Protein A, Protein G, ananti-constant region (light chain or heavy chain) antibody, and ananti-idiotype antibody.

Although the disclosed antigen binding molecules were produced in arabbit system, human, partially human, or humanized antibodies may besuitable for many applications, particularly those involvingadministration of the antibody to a human subject, other types ofantigen binding molecules will be suitable for certain applications.Such antibodies can be prepared as described herein and form an aspectof the instant disclosure.

The instant disclosure provides antigen binding molecules thatspecifically bind to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules. Antigenbinding molecules that cross compete with the disclosed antigen bindingmolecules disclosed herein for an aspect of the disclosure. In certainembodiments, the antigen binding molecule cross competes with areference antibody comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-60. In certain embodiments, theantigen binding molecule cross competes with a reference antibody,wherein the reference antibody comprises a VH CDR1 comprising an aminoacid sequence of SEQ ID NOs: 5, 23, 41 and 53. In certain embodiments,the antigen binding molecule cross competes with a reference antibody,wherein the reference antibody comprises a VH CDR2 comprising an aminoacid sequence of SEQ ID NOs: 6, 24, 42 and 54. In certain embodiments,the antigen binding molecule cross competes with a reference antibody,wherein the reference antibody comprises a VH CDR3 comprising an aminoacid sequence of SEQ ID NOs: 7, 25, 43 and 55. In certain embodiments,the antigen binding molecule cross competes with a reference antibody,wherein the reference antibody comprises a VL CDR1 comprising an aminoacid sequence of SEQ ID NOs: 11, 29, 47 and 59. In certain embodiments,the antigen binding molecule cross competes with a reference antibody,wherein the reference antibody comprises a VL CDR2 comprising an aminoacid sequence of SEQ ID NOs: 12, 30, 48 and 60. In certain embodiments,the antigen binding molecule cross competes with a reference antibody,wherein the reference antibody comprises a VL CDR3 comprising an aminoacid sequence of SEQ ID NOs: 13, 31, 49 and 61.

In some embodiments, the polynucleotides of the present inventionencodes an antibody or antigen binding molecule that specifically bindsthe anti-CD19 scFv FMC63 (SEQ ID NO: 1), as well as molecules comprisingthese sequences and cells presenting such molecules, wherein theantibody or antigen binding molecule binds the same or an overlappingepitope as a reference antibody disclosed herein (e.g., those comprisingsequences presented in FIGS. 5-21). In certain embodiments, the antibodyor antigen binding molecule binds the same or an overlapping epitope asa reference antibody.

II.A. Clone 7

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence GFSFNN (SEQ ID NO: 5).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence YVGSSD (SEQ ID NO: 6).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence NLGL (SEQ ID NO: 7).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa heavy chain VH comprising: (a) a VH CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence GFSFNN (SEQ ID NO: 5);and/or (b) a VH CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence YVGSSD (SEQ ID NO: 6); and/or (c)a VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence NLGL (SEQ ID NO: 7).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VHCDR3 comprise the amino acid sequence of the VH CDR1, VH CDR2, and VHCDR3 sequences presented in FIGS. 5, 19, 20 and 21. In a particularembodiment, the VH CDRs are those presented in FIG. 5.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence comprising an amino acid sequenceof FIG. 5 (SEQ ID NO: 3).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VH framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VHFRs as set forth in, or derivable from, the sequences presented in FIG.5 (e.g., one, two, three, or four of the FRs in one sequence of FIG. 5).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., SEQ ID NO: 4 in FIG. 5). Insome embodiments, the antibody or antigen binding molecule comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 3.

In various embodiments, the heavy chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theheavy chain variable region sequence of SEQ ID NO:3.

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence QASESVYNSDWLA (SEQ ID NO: 11).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence AASTLAS (SEQ ID NO: 12).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence AGYKSSSTDGIA (SEQ ID NO: 13).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa light chain VL comprising: (a) a VL CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence QASESVYNSDWLA (SEQ IDNO: 11); and/or (b) a VL CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence AASTLAS (SEQ ID NO: 12); and/or(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence AGYKSSSTDGIA (SEQ ID NO: 13).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVL CDR1, a VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VLCDR3 comprise the amino acid sequence of the VL CDR1, VL CDR2, and VLCDR3 sequences presented in FIGS. 6, 19, 20 and 21. In a particularembodiment, the VL CDRs are those presented in FIG. 6.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain variable region sequence comprising an amino acid sequenceof FIG. 6 (SEQ ID NO: 9)

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VL framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VLFRs as set forth in, or derivable from, the sequences presented in FIG.6 (e.g., one, two, three, or four of the FRs in one sequence of FIG. 6).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain sequence disclosed herein (e.g., SEQ ID NO: 10 in FIG. 6).In some embodiments, the antibody or antigen binding molecule comprisesa light chain variable region comprising the amino acid sequence of SEQID NO: 9.

In various embodiments, the light chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain variable region sequence of SEQ ID NO: 9.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesany one, two, and/or three VH CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VHCDR1, a VH CDR2, and a VH CDR3 having the amino acid sequence of any VHCDR1, VH CDR2, and VH CDR3 disclosed herein, respectively. In someembodiments, the antibody or antigen binding molecule comprises any one,two, and/or three VL CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VLCDR1, a VL CDR2, and a VL CDR3 having the amino acid sequence of any VLCDR1, VL CDR2, and VL CDR3 disclosed herein, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region comprising the amino acid sequence of SEQ ID NO: 5;(b) a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 6;(c) a VH CDR3 region comprising the amino acid sequence of SEQ ID NO: 7;(d) a VL CDR1 region comprising the amino acid sequence of SEQ ID NO:11; (e) a VL CDR2 region comprising the amino acid sequence of SEQ IDNO: 12; and (f) a VL CDR3 region comprising the amino acid sequence ofSEQ ID NO: 13.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region; (b) a VH CDR2 region; (c) a VH CDR3 region; (d) aVL CDR1 region; (e) a VL CDR2 region; and (f) a VL CDR3 region, whereinthe VH and VL CDRs are shown in FIGS. 5, 6, respectively, and in FIGS.19, 20 and 21.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence disclosed herein (e.g., in FIG. 5)and a light chain variable region sequence disclosed herein (e.g., inFIG. 6).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 3; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 9. Nucleotide sequences encoding the heavychain variable region and the light chain variable region are providedin FIGS. 5 and 6, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., in FIG. 5) and a lightchain sequence disclosed herein (e.g., in FIG. 6).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 4;and (b) a light chain comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising an amino acid sequence that is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 4; and (b) a light chain comprising anamino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10.

II.B. Clone 13

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence GFSFNN (SEQ ID NO: 5).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence YVGSSD (SEQ ID NO: 6).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence NLGL (SEQ ID NO: 7).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa heavy chain VH comprising: (a) a VH CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence GFSFNN (SEQ ID NO: 5);and/or (b) a VH CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence YVGSSD (SEQ ID NO: 6); and/or (c)a VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence NLGL (SEQ ID NO: 7).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VHCDR3 comprise the amino acid sequence of the VH CDR1, VH CDR2, and VHCDR3 sequences presented in FIGS. 7, 19, 20 and 21. In a particularembodiment, the VH CDRs are those presented in FIG. 7.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence comprising an amino acid sequenceof FIG. 7 (SEQ ID NO: 15).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VH framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VHFRs as set forth in, or derivable from, the sequences presented in FIG.7 (e.g., one, two, three, or four of the FRs in one sequence of FIG. 7).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., SEQ ID NO: 16 in FIG. 7).In some embodiments, the antibody or antigen binding molecule comprisesa heavy chain variable region comprising the amino acid sequence of SEQID NO: 15.

In various embodiments, the heavy chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theheavy chain variable region sequence of SEQ ID NO:15.

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence QASESVYNSDWLA (SEQ ID NO: 11).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence AASTLAS (SEQ ID NO: 12).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence AGYKSSSTDGIA (SEQ ID NO: 13).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa light chain VL comprising: (a) a VL CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence QASESVYNSDWLA (SEQ IDNO: 11); and/or (b) a VL CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence AASTLAS (SEQ ID NO: 12); and/or(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence AGYKSSSTDGIA (SEQ ID NO: 13).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVL CDR1, a VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VLCDR3 comprise the amino acid sequence of the VL CDR1, VL CDR2, and VLCDR3 sequences presented in FIGS. 8, 19, 20 and 21. In a particularembodiment, the VL CDRs are those presented in FIG. 8.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain variable region sequence comprising an amino acid sequenceof FIG. 8 (SEQ ID NO: 18)

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VL framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VLFRs as set forth in, or derivable from, the sequences presented in FIG.8 (e.g., one, two, three, or four of the FRs in one sequence of FIG. 8).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain sequence disclosed herein (e.g., SEQ ID NO: 19 in FIG. 8).In some embodiments, the antibody or antigen binding molecule comprisesa light chain variable region comprising the amino acid sequence of SEQID NO: 18.

In various embodiments, the light chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain variable region sequence of SEQ ID NO: 18.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesany one, two, and/or three VH CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VHCDR1, a VH CDR2, and a VH CDR3 having the amino acid sequence of any VHCDR1, VH CDR2, and VH CDR3 disclosed herein, respectively. In someembodiments, the antibody or antigen binding molecule comprises any one,two, and/or three VL CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VLCDR1, a VL CDR2, and a VL CDR3 having the amino acid sequence of any VLCDR1, VL CDR2, and VL CDR3 disclosed herein, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region comprising the amino acid sequence of SEQ ID NO: 5;(b) a VH CDR2 region comprising the amino acid sequence of SEQ ID NO: 6;(c) a VH CDR3 region comprising the amino acid sequence of SEQ ID NO: 7;(d) a VL CDR1 region comprising the amino acid sequence of SEQ ID NO:11; (e) a VL CDR2 region comprising the amino acid sequence of SEQ IDNO: 12; and (f) a VL CDR3 region comprising the amino acid sequence ofSEQ ID NO: 13.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region; (b) a VH CDR2 region; (c) a VH CDR3 region; (d) aVL CDR1 region; (e) a VL CDR2 region; and (f) a VL CDR3 region, whereinthe VH and VL CDRs are shown in FIGS. 7, 8, respectively, and in FIGS.19, 20 and 21.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence disclosed herein (e.g., in FIG. 7)and a light chain variable region sequence disclosed herein (e.g., inFIG. 8).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 15; and (b) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 18. Nucleotide sequences encoding theheavy chain variable region and the light chain variable region areprovided in FIGS. 7 and 8, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., in FIG. 7) and a lightchain sequence disclosed herein (e.g., in FIG. 8).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 16;and (b) a light chain comprising the amino acid sequence of SEQ ID NO:19.

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising an amino acid sequence that is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 16; and (b) a light chain comprisingan amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:19.

II.C. Clone 14-1

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence GFDFSINY (SEQ ID NO: 23).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence YTGDD (SEQ ID NO: 24).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence GLYSGSINNL (SEQ ID NO: 25).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa heavy chain VH comprising: (a) a VH CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence GFDFSINY (SEQ ID NO:23); and/or (b) a VH CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence YTGDD (SEQ ID NO: 24); and/or (c)a VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence GLYSGSINNL (SEQ ID NO: 25).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VHCDR3 comprise the amino acid sequence of the VH CDR1, VH CDR2, and VHCDR3 sequences presented in FIGS. 19, 20, and 21. In a particularembodiment, the VH CDRs are those presented in FIG. 9.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence comprising an amino acid sequenceof FIG. 9 (SEQ ID NO: 21).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VH framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VHFRs as set forth in, or derivable from, the sequences presented in FIG.9 (e.g., one, two, three, or four of the FRs in one sequence of FIG. 9).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., SEQ ID NO: 22 in FIG. 9).In some embodiments, the antibody or antigen binding molecule comprisesa heavy chain variable region comprising the amino acid sequence of SEQID NO: 21.

In various embodiments, the heavy chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theheavy chain variable region sequence of SEQ ID NO:21.

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence QASQSVYNNDYLS (SEQ ID NO: 29).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence YASTLAS (SEQ ID NO: 30).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence AGVKGYSNDNNG (SEQ ID NO: 31).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa light chain VL comprising: (a) a VL CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence QASQSVYNNDYLS (SEQ IDNO: 29); and/or (b) a VL CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence YASTLAS (SEQ ID NO: 30); and/or(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence AGVKGYSNDNNG (SEQ ID NO: 31).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVL CDR1, a VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VLCDR3 comprise the amino acid sequence of the VL CDR1, VL CDR2, and VLCDR3 sequences presented in FIGS. 19, 20 and 21. In a particularembodiment, the VL CDRs are those presented in FIG. 10.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain variable region sequence comprising an amino acid sequenceof FIG. 10 (SEQ ID NO: 27).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VL framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VLFRs as set forth in, or derivable from, the sequences presented in FIG.10 (e.g., one, two, three, or four of the FRs in one sequence of FIG.10).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain sequence disclosed herein (e.g., SEQ ID NO: 28 in FIG. 10).In some embodiments, the antibody or antigen binding molecule comprisesa light chain variable region comprising the amino acid sequence of SEQID NO: 27.

In various embodiments, the light chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain variable region sequence of SEQ ID NO: 27.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesany one, two, and/or three VH CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VHCDR1, a VH CDR2, and a VH CDR3 having the amino acid sequence of any VHCDR1, VH CDR2, and VH CDR3 disclosed herein, respectively. In someembodiments, the antibody or antigen binding molecule comprises any one,two, and/or three VL CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VLCDR1, a VL CDR2, and a VL CDR3 having the amino acid sequence of any VLCDR1, VL CDR2, and VL CDR3 disclosed herein, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region comprising the amino acid sequence of SEQ ID NO:23; (b) a VH CDR2 region comprising the amino acid sequence of SEQ IDNO: 24; (c) a VH CDR3 region comprising the amino acid sequence of SEQID NO: 25; (d) a VL CDR1 region comprising the amino acid sequence ofSEQ ID NO: 29; (e) a VL CDR2 region comprising the amino acid sequenceof SEQ ID NO: 30; and (f) a VL CDR3 region comprising the amino acidsequence of SEQ ID NO: 31.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region; (b) a VH CDR2 region; (c) a VH CDR3 region; (d) aVL CDR1 region; (e) a VL CDR2 region; and (f) a VL CDR3 region, whereinthe VH and VL CDRs are shown in FIGS. 9, 10, respectively, and in FIGS.19, 20 and 21.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence disclosed herein (e.g., in FIG. 9)and a light chain variable region sequence disclosed herein (e.g., inFIG. 10).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 22; and (b) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 28. Nucleotide sequences encoding theheavy chain variable region and the light chain variable region areprovided in FIGS. 9 and 10, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., in FIG. 9) and a lightchain sequence disclosed herein (e.g., in FIG. 10).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 22;and (b) a light chain comprising the amino acid sequence of SEQ ID NO:28.

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising an amino acid sequence that is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 22; and (b) a light chain comprisingan amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:28.

II.D. Clone 14-7

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence GFDFSINY (SEQ ID NO: 23).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence YTGDD (SEQ ID NO: 24).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence GLYSGSINNL (SEQ ID NO: 25).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa heavy chain VH comprising: (a) a VH CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence GFDFSINY (SEQ ID NO:23); and/or (b) a VH CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence YTGDD (SEQ ID NO: 24); and/or (c)a VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence GLYSGSINNL (SEQ ID NO: 25).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VHCDR3 comprise the amino acid sequence of the VH CDR1, VH CDR2, and VHCDR3 sequences presented in FIGS. 19, 20 and 21. In a particularembodiment, the VH CDRs are those presented in FIG. 11.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence comprising an amino acid sequenceof FIG. 11 (SEQ ID NO: 33).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VH framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VHFRs as set forth in, or derivable from, the sequences presented in FIG.11 (e.g., one, two, three, or four of the FRs in one sequence of FIG.11).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., in FIG. 11). In someembodiments, the antibody or antigen binding molecule comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:33.

In various embodiments, the heavy chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theheavy chain variable region sequence of SEQ ID NO: 33.

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence QASQSVYNNDYLS (SEQ ID NO: 29).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence YASTLAS (SEQ ID NO: 30).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence AGVKGYSNDNNG (SEQ ID NO: 31).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa light chain VL comprising: (a) a VL CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence QASQSVYNNDYLS (SEQ IDNO: 29); and/or (b) a VL CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence YASTLAS (SEQ ID NO: 30); and/or(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence AGVKGYSNDNNG (SEQ ID NO: 31).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVL CDR1, a VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VLCDR3 comprise the amino acid sequence of the VL CDR1, VL CDR2, and VLCDR3 sequences presented in FIGS. 20, 21 and 22. In a particularembodiment, the VL CDRs are those presented in FIG. 12.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain variable region sequence comprising an amino acid sequenceof FIG. 12 (SEQ ID NO: 36).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VL framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VLFRs as set forth in, or derivable from, the sequences presented in FIG.12 (e.g., one, two, three, or four of the FRs in one sequence of FIG.12).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain sequence disclosed herein (e.g., SEQ ID NO: 37 in FIG. 12).In some embodiments, the antibody or antigen binding molecule comprisesa light chain variable region comprising the amino acid sequence of SEQID NO: 36.

In various embodiments, the light chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain variable region sequence of SEQ ID NO: 36.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesany one, two, and/or three VH CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VHCDR1, a VH CDR2, and a VH CDR3 having the amino acid sequence of any VHCDR1, VH CDR2, and VH CDR3 disclosed herein, respectively. In someembodiments, the antibody or antigen binding molecule comprises any one,two, and/or three VL CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VLCDR1, a VL CDR2, and a VL CDR3 having the amino acid sequence of any VLCDR1, VL CDR2, and VL CDR3 disclosed herein, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region comprising the amino acid sequence of SEQ ID NO:23; (b) a VH CDR2 region comprising the amino acid sequence of SEQ IDNO: 24; (c) a VH CDR3 region comprising the amino acid sequence of SEQID NO: 25; (d) a VL CDR1 region comprising the amino acid sequence ofSEQ ID NO: 29; (e) a VL CDR2 region comprising the amino acid sequenceof SEQ ID NO: 30; and (f) a VL CDR3 region comprising the amino acidsequence of SEQ ID NO: 31.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region; (b) a VH CDR2 region; (c) a VH CDR3 region; (d) aVL CDR1 region; (e) a VL CDR2 region; and (f) a VL CDR3 region, whereinthe VH and VL CDRs are shown in FIGS. 11, 12, respectively, and in FIGS.19, 20 and 21.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence disclosed herein (e.g., in FIG. 11)and a light chain variable region sequence disclosed herein (e.g., inFIG. 12).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 33; and (b) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 36. Nucleotide sequences encoding theheavy chain variable region and the light chain variable region areprovided in FIGS. 11 and 12, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., in FIG. 11) and a lightchain sequence disclosed herein (e.g., in FIG. 12).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 34;and (b) a light chain comprising the amino acid sequence of SEQ ID NO:37.

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising an amino acid sequence that is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 34; and (b) a light chain comprisingan amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:37.

II.E. Clone 15 (15-7)

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence GFSFTSNY (SEQ ID NO: 41).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence FLGSSG (SEQ ID NO: 42).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence DYVNGYDYFNL (SEQ ID NO: 43).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa heavy chain VH comprising: (a) a VH CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence GFSFTSNY (SEQ ID NO:41); and/or (b) a VH CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence FLGSSG (SEQ ID NO: 42); and/or(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence DYVNGYDYFNL (SEQ ID NO: 43).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VHCDR3 comprise the amino acid sequence of the VH CDR1, VH CDR2, and VHCDR3 sequences presented in FIGS. 19, 20 and 21. In a particularembodiment, the VH CDRs are those presented in FIG. 13.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence comprising an amino acid sequenceof FIG. 13 (SEQ ID NO: 39).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VH framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VHFRs as set forth in, or derivable from, the sequences presented in FIG.13 (e.g., one, two, three, or four of the FRs in one sequence of FIG.13).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., SEQ ID NO: 40 in FIG. 13).In some embodiments, the antibody or antigen binding molecule comprisesa heavy chain variable region comprising the amino acid sequence of SEQID NO: 39.

In various embodiments, the heavy chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theheavy chain variable region sequence of SEQ ID NO:39.

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence QSSQSVYNKNLA (SEQ ID NO: 47).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence STSTLDS (SEQ ID NO: 48).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence LGSYDCSSADCNA (SEQ ID NO: 49).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa light chain VL comprising: (a) a VL CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence QSSQSVYNKNLA (SEQ IDNO: 47); and/or (b) a VL CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence STSTLDS (SEQ ID NO: 48); and/or(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence LGSYDCSSADCNA (SEQ ID NO: 49).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVL CDR1, a VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VLCDR3 comprise the amino acid sequence of the VL CDR1, VL CDR2, and VLCDR3 sequences presented in FIGS. 19, 20 and 21. In a particularembodiment, the VL CDRs are those presented in FIG. 14.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain variable region sequence comprising an amino acid sequenceof FIG. 14 (SEQ ID NO: 45).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VL framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VLFRs as set forth in, or derivable from, the sequences presented in FIG.14 (e.g., one, two, three, or four of the FRs in one sequence of FIG.14).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain sequence disclosed herein (e.g., SEQ ID NO: 46 in FIG. 14).In some embodiments, the antibody or antigen binding molecule comprisesa light chain variable region comprising the amino acid sequence of SEQID NO: 45.

In various embodiments, the light chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain variable region sequence of SEQ ID NO: 45.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesany one, two, and/or three VH CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VHCDR1, a VH CDR2, and a VH CDR3 having the amino acid sequence of any VHCDR1, VH CDR2, and VH CDR3 disclosed herein, respectively. In someembodiments, the antibody or antigen binding molecule comprises any one,two, and/or three VL CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VLCDR1, a VL CDR2, and a VL CDR3 having the amino acid sequence of any VLCDR1, VL CDR2, and VL CDR3 disclosed herein, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region comprising the amino acid sequence of SEQ ID NO:41; (b) a VH CDR2 region comprising the amino acid sequence of SEQ IDNO: 42; (c) a VH CDR3 region comprising the amino acid sequence of SEQID NO: 43; (d) a VL CDR1 region comprising the amino acid sequence ofSEQ ID NO: 47; (e) a VL CDR2 region comprising the amino acid sequenceof SEQ ID NO: 48; and (f) a VL CDR3 region comprising the amino acidsequence of SEQ ID NO: 49.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region; (b) a VH CDR2 region; (c) a VH CDR3 region; (d) aVL CDR1 region; (e) a VL CDR2 region; and (f) a VL CDR3 region, whereinthe VH and VL CDRs are shown in FIGS. 13, 14, respectively, and in FIGS.19, 20 and 21.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence disclosed herein (e.g., in FIG. 13)and a light chain variable region sequence disclosed herein (e.g., inFIG. 14).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 39; and (b) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 45. Nucleotide sequences encoding theheavy chain variable region and the light chain variable region areprovided in FIGS. 13 and 14, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., in FIG. 13) and a lightchain sequence disclosed herein (e.g., in FIG. 14).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 40;and (b) a light chain comprising the amino acid sequence of SEQ ID NO:46.

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising an amino acid sequence that is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 40; and (b) a light chain comprisingan amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:46.

II.F. Clone 17

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence GFSFSDSW (SEQ ID NO: 53).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence YTGDG (SEQ ID NO: 54).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VH CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence GAQFYL (SEQ ID NO: 55).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa heavy chain VH comprising: (a) a VH CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence GFSFTSNY (SEQ ID NO:53); and/or (b) a VH CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence FLGSSG (SEQ ID NO: 54); and/or(c) a VH CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence DYVNGYDYFNL (SEQ ID NO: 55).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VHCDR3 comprise the amino acid sequence of the VH CDR1, VH CDR2, and VHCDR3 sequences presented in FIGS. 19, 20 and 21. In a particularembodiment, the VH CDRs are those presented in FIG. 15.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence comprising an amino acid sequenceof FIG. 15 (SEQ ID NO: 51).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VH framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VHFRs as set forth in, or derivable from, the sequences presented in FIG.15 (e.g., one, two, three, or four of the FRs in one sequence of FIG.15).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., SEQ ID NO: 52 in FIG. 15).In some embodiments, the antibody or antigen binding molecule comprisesa heavy chain variable region comprising the amino acid sequence of SEQID NO: 51.

In various embodiments, the heavy chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theheavy chain variable region sequence of SEQ ID NO:51.

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR1 comprising, consisting of, or consisting essentially of theamino acid sequence QSSQSVYANTYLS (SEQ ID NO: 59).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR2 comprising, consisting of, or consisting essentially of theamino acid sequence SASSLAS (SEQ ID NO: 60).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa VL CDR3 comprising, consisting of, or consisting essentially of theamino acid sequence LGRYSCGLADCAA (SEQ ID NO: 61).

In some embodiments, an antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting such molecules, comprisesa light chain VL comprising: (a) a VL CDR1 comprising, consisting of, orconsisting essentially of the amino acid sequence QSSQSVYANTYLS (SEQ IDNO: 59); and/or (b) a VL CDR2 comprising, consisting of, or consistingessentially of the amino acid sequence SASSLAS (SEQ ID NO: 60); and/or(c) a VL CDR3 comprising, consisting of, or consisting essentially ofthe amino acid sequence LGRYSCGLADCAA (SEQ ID NO: 61).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aVL CDR1, a VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VLCDR3 comprise the amino acid sequence of the VL CDR1, VL CDR2, and VLCDR3 sequences presented in FIGS. 20, 21 and 22. In a particularembodiment, the VL CDRs are those presented in FIG. 21.

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain variable region sequence comprising an amino acid sequenceof FIG. 16 (SEQ ID NO: 57).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesthe VL framework regions (FRs) described herein. In specificembodiments, the antibody or antigen binding molecule comprises the VLFRs as set forth in, or derivable from, the sequences presented in FIG.16 (e.g., one, two, three, or four of the FRs in one sequence of FIG.16).

In some embodiments, the antigen binding molecule or antibody thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises alight chain sequence disclosed herein (e.g., SEQ ID NO: 58 in FIG. 16).In some embodiments, the antibody or antigen binding molecule comprisesa light chain variable region comprising the amino acid sequence of SEQID NO: 57.

In various embodiments, the light chain variable region is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain variable region sequence of SEQ ID NO:57.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprisesany one, two, and/or three VH CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VHCDR1, a VH CDR2, and a VH CDR3 having the amino acid sequence of any VHCDR1, VH CDR2, and VH CDR3 disclosed herein, respectively. In someembodiments, the antibody or antigen binding molecule comprises any one,two, and/or three VL CDR sequences disclosed herein. In certainembodiments, the antibody or antigen binding molecule comprises a VLCDR1, a VL CDR2, and a VL CDR3 having the amino acid sequence of any VLCDR1, VL CDR2, and VL CDR3 disclosed herein, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region comprising the amino acid sequence of SEQ ID NO:53; (b) a VH CDR2 region comprising the amino acid sequence of SEQ IDNO: 54; (c) a VH CDR3 region comprising the amino acid sequence of SEQID NO: 55; (d) a VL CDR1 region comprising the amino acid sequence ofSEQ ID NO: 59; (e) a VL CDR2 region comprising the amino acid sequenceof SEQ ID NO: 60; and (f) a VL CDR3 region comprising the amino acidsequence of SEQ ID NO: 61.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises:(a) a VH CDR1 region; (b) a VH CDR2 region; (c) a VH CDR3 region; (d) aVL CDR1 region; (e) a VL CDR2 region; and (f) a VL CDR3 region, whereinthe VH and VL CDRs are shown in FIGS. 15, 16, respectively, and in FIGS.19, 20 and 21.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain variable region sequence disclosed herein (e.g., in FIG. 15)and a light chain variable region sequence disclosed herein (e.g., inFIG. 16).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 45; and (b) a light chain variable region comprising theamino acid sequence of SEQ ID NO: 57. Nucleotide sequences encoding theheavy chain variable region and the light chain variable region areprovided in FIGS. 15 and 16, respectively.

In some embodiments, the antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, comprises aheavy chain sequence disclosed herein (e.g., in FIG. 15) and a lightchain sequence disclosed herein (e.g., in FIG. 16).

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 52;and (b) a light chain comprising the amino acid sequence of SEQ ID NO:58.

In some embodiments, the antibody or antigen binding molecule comprises:(a) a heavy chain comprising an amino acid sequence that is 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 52; and (b) a light chain comprisingan amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:58.

III Polynucleotides Encoding Antibodies and Other Antigen BindingMolecules

The present invention is also directed to polynucleotides encodingantibodies and other antigen binding molecules that specifically bind tothe anti-CD19 scFv FMC63 (SEQ ID NO: 1), molecules comprising thissequence and cells presenting this sequence.

In some embodiments, a polynucleotide of the present invention encodesan antigen binding molecule, wherein the antigen binding moleculecomprises a heavy chain variable region amino acid sequence that is atleast about 75%, at least about 85%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% identical to a heavy chainvariable region amino acid sequence selected from the group consistingof SEQ ID NOs: 3, 15, 21, 33, 39 and 51.

In some embodiments, a polynucleotide of the present invention encodesantigen binding molecule, wherein the antigen binding molecule comprisesa light chain variable amino acid sequence that is at least about 75%,at least about 85%, at least about 85%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or 100% identical to a light chain variable regionamino acid sequence selected from the group consisting of SEQ ID NOs: 8,18, 27, 36, 45 and 57.

In certain embodiments, the polynucleotide comprises a heavy chaincoding sequence selected from the group consisting of SEQ ID NO: 2, SEQID NO: 14, SEQ ID NO: 20, SEQ ID NO: 32, SEQ ID NO: 38 and SEQ ID NO:50. In another embodiment, the polynucleotide comprises a light chaincoding sequence selected from the group consisting of SEQ ID NO: 8, SEQID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 44 and SEQ ID NO:56.

As will be appreciated by those of skill in the art, variations of thedisclosed polynucleotide sequences are possible due to the degeneracy ofthe genetic code. Such variants of the disclose polynucleotide sequencesthus form an aspect of the instant disclosure.

IV. Vectors, Cells, and Pharmaceutical Compositions

In certain aspects, provided herein are vectors comprising apolynucleotide disclosed herein. In some embodiments, the presentinvention is directed to a vector or a set of vectors comprising apolynucleotide(s) encoding an amino acid sequence of an antibody orantigen binding molecule that specifically binds to the anti-CD19 scFvFMC63 (SEQ ID NO: 1), molecules comprising this sequence and cellspresenting this sequence, as described herein.

Any vector known in the art can be suitable for expressing theantibodies and other antigen binding molecules of the present invention.In some embodiments, the vector is a viral vector. In some embodiments,the vector is a retroviral vector, a DNA vector, a murine leukemia virusvector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, abaculoviral vector, an Epstein Barr viral vector, a papovaviral vector,a vaccinia viral vector, a herpes simplex viral vector, an adenovirusassociated vector (AAV), a lentiviral vector, or any combinationthereof.

In other aspects, provided herein are cells comprising a polynucleotideor a vector of the present invention. In some embodiments, the presentinvention is directed to cells, in vitro cells, comprising apolynucleotide encoding an antigen binding molecule, as describedherein. In some embodiments, the present invention is directed to cells,e.g., in vitro cells, comprising a polynucleotide encoding an antibodyor an antigen binding molecule thereof that specifically binds to theanti-CD19 scFv FMC63 (SEQ ID NO: 1), molecules comprising this sequenceand cells presenting this sequence, as disclosed herein.

Any cell can be used as a host cell for the polynucleotides and vectorsencoding all or a fragment of the antibodies and other antigen bindingmolecules of the present invention. In some embodiments, a host cell canbe a prokaryotic cell, fungal cell, yeast cell, or higher eukaryoticcells such as a mammalian cell. Suitable prokaryotic cells include,without limitation, eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, Enterobactehaceae such as Escherichia, e.g., E.coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g.,Salmonella typhimurium; Serratia, e.g., Serratia marcescans, andShigella; Bacilli such as B. subtilis and B. licheniformis; Pseudomonassuch as P. aeruginosa; and Streptomyces. In some embodiments, a hostcell is a human cell. In some embodiments, a host cell is a CHO cell andin other embodiments a host cell is a sP2/0 or other murine cell. A hostcell of the present invention can be obtained through any source knownin the art.

Other aspects of the present invention are directed to compositionscomprising a polynucleotide described herein, a vector described herein,an antibody and/or an antigen binding molecule described herein, or anin vitro cell described herein. In some embodiments, the compositioncomprises a pharmaceutically acceptable carrier, diluent, solubilizer,emulsifier, preservative and/or adjuvant. In some embodiments, thecomposition comprises an excipient. In some embodiments, the compositioncomprises a polynucleotide encoding an antibody or antigen bindingmolecule that specifically binds to that specifically binds to theanti-CD19 scFv FMC63 (SEQ ID NO: 1), molecules comprising this sequenceand cells presenting this sequence. In another embodiment, thecomposition comprises an antigen binding molecule encoded by apolynucleotide of the present invention, wherein the antigen bindingmolecule specifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1),molecules comprising this sequence and cells presenting this sequence,as disclosed herein. In another embodiment, the composition comprises anin vitro cell comprising a polynucleotide encoding an antibody or anantigen binding molecule thereof encoded by a polynucleotide of thepresent invention.

In some embodiments, the composition comprises one antibody or antigenbinding molecule that specifically binds to the anti-CD19 scFv FMC63(SEQ ID NO: 1), molecules comprising this sequence and cells presentingthis sequence, as disclosed herein. In some embodiments, the compositioncomprises more than one antibody or antigen binding molecule thatspecifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1), moleculescomprising this sequence and cells presenting this sequence, asdisclosed herein, wherein the antibodies or antigen binding moleculesbind more than one epitope. In some embodiments, the antibodies orantigen binding molecules will not compete with one another for bindingto that epitope. In some embodiments, two or more of the antibodies orantigen binding molecules provided herein are combined together in apharmaceutical composition. Preferably such a composition will besuitable for administration to a subject, including a human.

V. Exemplary Methods

The following section describes various exemplary methods of using thedisclosed antigen binding molecules herein. Any antigen binding moleculedisclosed herein can be employed in the disclosed methods.

In various embodiments of the disclosed methods, the antigen bindingmolecule is selected from the group consisting of an antibody, an scFv,a Fab, a Fab′, a Fv, a F(ab′)₂, a dAb, a human antibody, a humanizedantibody, a chimeric antibody, a monoclonal antibody, a polyclonalantibody, a recombinant antibody, an IgE antibody, an IgD antibody, anIgM antibody, an IgG1 antibody, an IgG1 antibody having at least onemutation in the hinge region, an IgG2 antibody an IgG2 antibody havingat least one mutation in the hinge region, an IgG3 antibody, an IgG1antibody having at least one mutation in the hinge region, an IgG4antibody, an IgG4 antibody having at least one mutation in the hingeregion, an antibody comprising at least one non-naturally occurringamino acid, and any combination thereof.

In some of the disclosed methods T cells can be employed. Such T cellscan come from any source known in the art. For example, T cells can bedifferentiated in vitro from a hematopoietic stem cell population, or Tcells can be obtained from a subject. T cells can be obtained from,e.g., peripheral blood mononuclear cells (PBMCs), bone marrow, lymphnode tissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. In addition, the Tcells can be derived from one or more T cell lines available in the art.T cells can also be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as FICOLL™ separation and/or apheresis. Additional methods ofisolating T cells for a T cell therapy are disclosed in U.S. PatentPublication No. 2013/0287748, which is herein incorporated by referencesin its entirety.

In various embodiments of the disclosed methods, an antigen bindingmolecule specifically binds to the anti-CD19 scFv FMC63 (SEQ ID NO: 1),molecules comprising this sequence and cells presenting this sequence,as disclosed herein. In further embodiments of the disclosed methods,the antigen binding molecule comprises one or more of (a) a light chainCDR1, (b) a light chain CDR2, (c) a light chain CDR3, (d) a heavy chainCDR1, (e) a heavy chain CDR2, and (f) a heavy chain CDR3. In additionalembodiments of the disclosed methods, an antigen binding moleculecomprises a heavy chain CDR3 comprising one of SEQ ID NOs: 7, 25, 43,55, or a light chain CDR3 comprising one of SEQ ID NOs: 13, 31, 49, 61,or both the heavy and light chain CDR3s. In other embodiments of thedisclosed methods, the antigen binding molecule comprises a heavy chainCDR1 comprising an amino acid sequence comprising one of SEQ ID NOs: 5,23, 41 and 53, or a heavy chain CDR2 comprising the amino acid sequenceof one of SEQ ID NOs: 6, 24, 42 and 54, or a light chain CDR1 comprisingthe amino acid sequence of one of SEQ ID NOs: 11, 29, 47 and 59 or alight chain CDR2 comprising the amino acid sequence of one of SEQ IDNOs: 12, 30, 48 and 60. Referring to the Figures, in various embodimentsof the disclosed methods, the antigen binding molecule comprises a heavychain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1,a light chain CDR2, and a light chain CDR3, each CDR comprising an aminoacid sequence shown in FIGS. 5-21.

In various embodiments of the disclosed methods, an antigen bindingmolecule comprises a heavy chain (HC), and the HC comprises a heavychain variable region (VH) sequence comprising one of SEQ ID NOs: 3, 15,21, 33, 39 and 51. Referring to the figures, in various embodiments ofthe disclosed methods the heavy chain comprises a heavy chain CDR1, aheavy chain CDR2, and a heavy chain CDR3, each CDR comprising an aminoacid sequence shown in FIGS. 5-21. Moreover, in embodiments of thedisclosed methods, an antigen binding molecule can be employed whichcomprises a VH amino acid sequence that is at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or about 100% identical to a VH of anantigen binding molecule of claim disclosed herein (e.g., an antigenbinding molecules comprising a variable region (VH) sequence comprisingone of SEQ ID NOs: 3, 15, 21, 33, 39 and 51).

In various embodiments of the disclosed methods, an antigen bindingmolecule comprises a light chain (LC), and the LC can comprise a heavychain variable region (VL) sequence comprising one of SEQ ID NOs: 9, 18,27, 36, 44 and 57. Referring to the figures, in various embodiments ofthe disclosed methods the light chain comprises a light chain CDR1, alight chain CDR2, and a light chain CDR3, each CDR comprising an aminoacid sequence shown in FIGS. 5-21. Moreover, in embodiments of thedisclosed methods, an antigen binding molecule can be employed whichcomprises a VL amino acid sequence that is at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or about 100% identical to a VL of anantigen binding molecule of claim disclosed herein (e.g., an antigenbinding molecules comprising a variable region (VL) sequence comprisingSEQ ID NO: 9, 18, 27, 36, 44 and 57).

In view of the above description of antigen binding molecules that canbe employed in the disclosed methods, representative methods will now bediscussed in more detail.

Va. Method of Administering a Dose of a Medicament to a Subject

In one aspect, a method of administering a dose of a medicament to asubject, the dose comprising a preselected number of cells presenting atherapeutic molecule comprising the anti-CD19 scFv FMC63 (SEQ ID NO: 1),is provided.

In specific embodiments, the dose comprises 0.5×10⁶ cells per kilogramof the subject, 1.0×10⁶ cells per kilogram of the subject, 2.0×10⁶ cellsper kilogram of the subject, 3.0×10⁶ cells per kilogram of the subject,4.0×10⁶ cells per kilogram of the subject, or 5.0×10⁶ cells per kilogramof the subject, although the method can be employed using any dose.1.0×10⁶ cells per kilogram of the subject is a preferred dose.

Consistent with the definition provided herein, in various embodiments,a subject is a human or non-human subject. When the subject is a human,the subject can be, e.g., any human who is being treated for an abnormalphysiological condition, such as cancer or has been formally diagnosedwith a disorder, those without formally recognized disorders, thosereceiving medical attention, those at risk of developing the disorders,those being studied for the presence or absence of a disorder, etc.

Initially, a sample of known volume comprising a population comprising aknown number of cells, which cells are known or suspected to bepresenting a molecule comprising SEQ ID NO: 1, is provided. In thedisclosed method, the number of cells can be determined using any knownmethod. In preferred embodiments the cells are counted using anautomated apparatus, such as a cell sorter (e.g., a FACS), howevertraditional non-automated cell counting methods can also be employed.

The cells of the method can comprise any type of cell, with immune cells(e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells,keratinocytes, endothelial cells, astrocytes, fibroblasts, andoligodendrocytes). T cells (including T cytotoxic, T helper and Tregcells) are especially preferred. In specific embodiments, the cells areT cells, which can be obtained as described herein and by methods knownin the art. Any type of cell can be employed in the method, and the cellcan be a human or non-human cell (including both prokaryotic andeukaryotic cells). Exemplary cells include, but are not limited toimmune cells such as T cells, tumor infiltrating lymphocytes (TILs), NKcells, TCR-expressing cells, dendritic cells, and NK-T cells. The Tcells can be autologous, allogeneic, or heterologous. In additionalembodiments, the cells are T cells presenting a CAR. The T cells can beCD4+ T cells or CD8+ T cells. When a T cell is employed in the disclosedmethods, the T cell can be an in vivo T cell or an in vitro T cell.Moreover, the cells can be disposed in, or isolated from, anyenvironment capable of maintaining the cells in a viable form, such asblood, tissue or any other sample obtained from a subject, cell culturemedia, tissue grown ex vivo, etc. Gradient purification, cell cultureselection and/or cell sorting can also be employed in obtaining T cells.

The therapeutic molecule expressed by the cell can comprise any moleculeknown or suspected to provide a therapeutic benefit to a subject towhich is it administered. Thus, a therapeutic molecule can be a peptideor polypeptide of any structure or design. Preferably the SEQ ID NO: 1component is expressed or disposed, at least in part, extracellularly,i.e., to a degree that it can be recognized by an extracellularinteraction partner such as the antigen binding molecules of the instantdisclosure.

In specific embodiments, the therapeutic molecule is a CAR. When thetherapeutic molecule is a CAR it can comprise a molecule, or fragmentthereof, selected from the group consisting of CD28, OX-40, 4-1BB/CD137,CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cellco-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1,CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276(B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gammareceptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulinprotein, cytokine receptor, integrins, Signaling Lymphocytic ActivationMolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE,CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof.

Continuing, an aliquot of the sample comprising a population of cellspresenting a molecule comprising the anti-CD19 scFv FMC63 sequence (SEQID NO: 1) is provided. The aliquot can be obtained using any convenientmeans, such as by a cell sorter, by a simply pipetting of material outof the sample, etc.

Additionally, an antigen binding molecule that specifically binds theanti-CD19 scFv FMC63 (SEQ ID NO: 1) further comprising a detectablelabel is provided. The antigen binding molecule is preferably an antigenbinding molecule disclosed herein, e.g., in the figures, sequencelisting or the instant disclosure. Any detectable label can be employedin the method, as described herein, and suitable labels can be selectedusing a desired set of criteria. Examples of types of detectable labelsinclude fluorescent labels (e.g., an Atto dye, an Alexafluor dye,quantum dots, Hydroxycoumarin, Aminocoumarin, Methoxycoumarin, CascadeBlue, Pacific Blue, Pacific Orange, Lucifer yellow, NBD, R-Phycoerythrin(PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed,FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7,TRITC, X-Rhodamine, Lissamine Rhodamine B, Texas Red, Allophycocyanin(APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP(Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv,T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66Wmutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation),Midorishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP,GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green,ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP,ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO,TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry). Suitable opticaldyes, including fluoro-phores, are described in Johnson, MolecularProbes Handbook: A Guide to Fluorescent Probes and Labeling Techniques,11^(th) Edition, Life Technologies, (2010), hereby expresslyincorporated by reference, radiolabels (e.g., isotope markers such as³H, ¹¹C, ¹⁴C, ¹⁵N, ¹⁸F, ³⁵S, ⁶⁴CU, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁴I, ¹²⁵I, ¹³¹I),photochromic compounds, a Halo-tag, Atto dyes, Tracy dyes, proteinaceousfluorescent labels (e.g., proteinaceous fluorescent labels also include,but are not limited to, green fluorescent protein, including a Renilla,Ptilosarcus, or Aequorea species of GFP (Chalfie et al., (1994) Science263:802-805), EGFP (Clon-tech Labs., Inc., Genbank Accession NumberU55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc;Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr.Biol. 6: 178-182), enhanced yellow fluorescent protein (Clontech Labs.,Inc.), luciferase (Ichiki et al., (1993) J. Immunol. 150:5408-5417),magnetic labels (e.g., DYNABEADS), etc. Strategies for the labeling ofproteins are known in the art and can be employed in the disclosedmethod.

The label can be associated with the antigen binding molecule at anyposition in the molecule, although it is preferable to associate thelabel with the molecule at a position (or positions, if multiple labelsare employed) at a point such that the binding properties of themolecule are not modified (unless such modified binding activity isdesired). Any antigen binding molecule that specifically binds SEQ IDNO: 1 (or fragment thereof) can be employed. Multiple examples ofsuitable antigen binding molecules are provided herein, e.g., thosehaving one or more of the CDRs shown in FIGS. 5-21.

The antigen binding molecule can be disposed on any surface, or nosurface at all. For example, the antigen binding molecule can be presentin a buffer and the buffer-antigen binding molecule can be contactedwith the sample. Alternatively, the antigen binding molecule can beassociated with a surface. Suitable surfaces include agarose beads,magnetic beads such as DYNABEADS, or a plastic, glass or ceramic platesuch as a welled plate, a bag such as a cell culture bag, etc. Thesurface can itself be disposed in another structure, such as a column.

Continuing, the aliquot of the sample is contacted with the antigenbinding molecule under conditions that permit the formation of a bindingcomplex comprising a cell present in the sample and the antigen bindingmolecule. Thus, the result of this step of the method is the formationof a binding complex in which the antigen binding molecule, with which adetectable label is associated, is bound to the cell expressing thetherapeutic molecule, which comprises the anti-CD19 scFv FMC63 sequence(SEQ ID NO: 1). Thus, the binding complex itself is detectable.Conditions that permit the formation of a binding complex will bedependent on a variety of factors, however generally aqueous buffers atphysiological pH and ionic strength, such as in phosphate-bufferedsaline (PBS), will favor formation of binding complexes and arepreferred in the disclosed method.

The fraction of cells present in a binding complex in the aliquot isthen determined. This calculation can be performed by comparing thenumber of cells bearing the detectable label to those that do not, andcan be represented as percentage. The number of cells in bindingcomplexes can be determined. The specific method employed to determinethe number of cells present in a binding complex will be dependent onthe nature of the label selected. For example, FACS can be employed whena fluorescent label is selected; when an isotope label is selected massspectrometry, NMR or other technique can be employed; magnetic-basedcell sorting can be employed when a magnetic label is chosen; microscopycan also be employed. The number of cells in the sample is known abinitio and thus the fraction of cells present in a binding complex canbe easily determined.

Continuing, the concentration of cells in the initial sample expressinga molecule comprising the anti-CD19 scFv FMC63 (SEQ ID NO: 1) isdetermined; the determination is based on the fraction of cellsdetermined to be present in the binding complex, and thus expressing thetherapeutic protein bearing a detectable label.

The fraction of cells presenting the therapeutic protein is known, andthe volume of the aliquot is known; thus a simple comparison of thenumber of cells in the sample from which the aliquot was taken that areexpressing the therapeutic molecule to the volume of the larger sampleprovides the fraction of the cells in the sample bearing the therapeuticmolecule on a therapeutic molecule/volume basis (i.e., the concentrationof cells bearing the therapeutic molecule in the larger sample).

The volume of the sample that comprises the selected number of cells isthen determined, by extrapolation based on the concentration of cellsbearing therapeutic molecule present in the sample.

Finally, the volume of sample comprising the desired number of cells isadministered to the subject. The administration can comprise an aspectof a therapeutic regimen based on the therapeutic molecule present inthe sample and expressed by the cells in the sample.

Although the administration can be performed one time or more than onetime, an advantage of the method is that by administering a dosecomprising the preselected number of cells, which number of cells willbe determined based on a known or expected efficacy, unnecessaryadministration of cells presenting the therapeutic molecule is avoided;i.e., the subject receives the correct number of cells to provide adesired therapeutic benefit and is not overdosed with cells.

Vb. Method of Determining a Number of Cells Presenting a Molecule ofInterest

There are situations in which it may be desirable to determine thenumber of cells present in a sample. For example, it may be desirable todetermine the number of immune cells present a sample obtained from asubject. Or it may be desirable to determine the number of cellstransfected and expressing a construct, which can be used as a measureof the level of efficiency of the transfection. The disclosed method canbe employed in these and other applications in which it is desirable todetermine the number of cells present in a sample.

Thus, a method of determining a number of cells presenting a molecule ina sample wherein the molecule comprising the anti-CD19 scFv FMC63 (SEQID NO: 1) is provided.

In one embodiment, a sample comprising cells known or suspected to beexpressing a molecule comprising the amino acid sequence of theanti-CD19 scFv FMC63 (SEQ ID NO: 1) is provided.

The cell can be of any type, and can be human or non-human (e.g., mouse,rate, rabbit, hamster, etc). In one preferred embodiment, the cell is animmune cell. An immune cell of the method can be any type of immune cell(e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells,keratinocytes, endothelial cells, astrocytes, fibroblasts, andoligodendrocytes). T cells (including T cytotoxic, T helper and Tregcells) are especially preferred. In specific embodiments, the cells areT cells, which can be obtained as described herein and by methods knownin the art. Any type of immune cell can be employed in this embodimentof the disclosed method, and the cell can be a human or non-human cell(including both prokaryotic and eukaryotic cells). Exemplary cellsinclude, but are not limited to immune cells such as T cells, tumorinfiltrating lymphocytes (TILs), NK cells, TCR-expressing cells,dendritic cells, and NK-T cells. The T cells can be autologous,allogeneic, or heterologous. In additional embodiments, the cells are Tcells presenting a CAR. The T cells can be CD4+ T cells or CD8+ T cells.When a T cell is employed in the disclosed methods, the T cell can be anin vivo T cell or an in vitro T cell. Moreover, the cells can bedisposed in, or isolated from, any environment capable of maintainingthe cells in a viable form, such as blood, tissue or any other sampleobtained from a subject, cell culture media, tissue grown ex vivo, asuitable buffer, etc.

In specific embodiments, the molecule comprising the anti-CD19 scFvFMC63 (SEQ ID NO: 1) is a CAR. When the molecule is a CAR it cancomprise a molecule, or fragment thereof, selected from the groupconsisting of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40,Programmed Death-1 (PD-1), inducible T cell co-stimulator (ICOS),lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT,(TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokinereceptor, integrins, Signaling Lymphocytic Activation Molecules (SLAMproteins), activating NK cell receptors, BTLA, a Toll ligand receptor,ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103,ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof.

The sample is then contacted with an antigen binding molecule thatspecifically binds SEQ ID NO: 1 and comprises a detectable label, underconditions that permit the formation of a binding complex comprising acell present in the sample and the antigen binding molecule. The antigenbinding molecule is preferably an antigen binding molecule (or fragmentthereof) disclosed herein, e.g., in the figures, sequence listing or theinstant section of the disclosure. Any antigen binding molecule thatspecifically binds SEQ ID NO: 1 can be employed in the disclosed method.Multiple examples of suitable antigen binding molecules are providedherein, e.g., those having one or more of the CDRs shown in FIG. 5-21.

Any detectable label can be employed in the method, as described herein,and suitable labels can be selected using a desired set of criteria.Examples of types of detectable labels include fluorescent labels (e.g.,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malachite green, stilbene, LuciferYellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640,Cy 5, Cy 5.5, LC Red 705, Oregon green, the Alexa-Fluor dyes (AlexaFluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, AlexaFluor 660, Alexa Fluor 680), Cascade Blue, Cas-cade Yellow andR-phycoerythrin (PE) (Molecular Probes), FITC, Rhodamine, and Texas Red(Pierce), Cy5, Cy5.5, Cy7 (Amersham Life Science). Suitable opticaldyes, including fluoro-phores, are described in Johnson, MolecularProbes Handbook: A Guide to Fluorescent Probes and Labeling Techniques,11^(th) Edition, Life Technologies, (2010), hereby expresslyincorporated by reference, radiolabels (e.g., isotope markers such as³H, ¹¹C, ¹⁴C, ¹⁵N, ¹⁸F, ³⁵S, ⁶⁴CU, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁴I, ¹²⁵I, ¹³¹I),photochromic compounds, a Halo-tag, Atto dyes, Tracy dyes, proteinaceousfluorescent labels (e.g., proteinaceous fluorescent labels also include,but are not limited to, green fluorescent protein, including a Renilla,Ptilosarcus, or Aequorea species of GFP (Chalfie et al., (1994) Science263:802-805), EGFP (Clon-tech Labs., Inc., Genbank Accession NumberU55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc;Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr.Biol. 6: 178-182), enhanced yellow fluorescent protein (Clontech Labs.,Inc.), luciferase (Ichiki et al., (1993) J. Immunol. 150:5408-5417),magnetic labels (e.g., DYNABEADS), etc. Strategies for the labeling ofproteins are well known in the art and can be employed in the disclosedmethod. See, e.g., Obermaier et al., (2015) Methods Mol Biol1295:153-65; Strack (2016) Nature Methods 13:33; Site-Specific ProteinLabeling: Methods and Protocols, (Gautier and Hinner, eds.) 2015,Springer.

The label can be associated with the antigen binding molecule at anyposition in the molecule, although it is preferable to associate thelabel with the molecule at a position (or positions, if multiple labelsare employed) at a point such that the binding properties of themolecule are not modified (unless such modified binding activity isdesired). Any antigen binding molecule that specifically binds theanti-CD19 scFv FMC63 sequence (SEQ ID NO: 1) (or fragment thereof) canbe employed, such as those disclosed herein, e.g., those having one ormore of the CDRs shown in FIGS. 5-21.

The antigen binding molecule can be disposed on any surface, or nosurface at all. For example, the antigen binding molecule can be presentin a buffer and the buffer-antigen binding molecule can be contactedwith the sample. Alternatively, the antigen binding molecule can beassociated with a surface. Suitable surfaces include agarose beads,magnetic beads such as DYNABEADS, or a plastic, glass or ceramic platesuch as a welled plate, a bag such as a cell culture bag, etc. Thesurface can itself be disposed in another structure, such as a column.

Conditions that permit the formation of a binding complex will bedependent on a variety of factors, however generally aqueous buffers atphysiological pH and ionic strength, such as in phosphate-bufferedsaline (PBS), will favor formation of binding complexes and arepreferred in the disclosed method.

Continuing, the number of cells present in a binding complex in thesample is determined. The specific method employed to determine thenumber of cells present in a binding complex will be dependent on thenature of the label selected. For example, FACS can be employed when afluorescent label is selected; when an isotope label is selected massspectrometry, NMR or other technique can be employed; magnetic-basedcell sorting can be employed when a magnetic label is chosen; microscopycan also be employed. The output of these detection methods can be inthe form of a number of cells or the output can be of a form that allowsthe calculation of the number of cells based on the output.

Vc. Method of Isolating a Molecule

It is of tremendous value to have the ability to separate differentpopulations of molecules, and particularly biologically-relevantmolecules, from one another. Using the antigen binding moleculesprovided herein, such separation can be achieved and employed in a rangeof biotechnological, biopharmaceutical and therapeutic applications.Thus, in one aspect of the instant disclosure, a method of isolating amolecule comprising the anti-CD19 scFv FMC63 (SEQ ID NO: 1) is provided.

In some embodiments, the method comprises providing a sample known orsuspected to comprise a molecule comprising the anti-CD19 scFv FMC63(SEQ ID NO: 1).

In specific embodiments, the molecule comprising the anti-CD19 scFvFMC63 (SEQ ID NO: 1) is a CAR. When the molecule is a CAR it cancomprise a molecule, or fragment thereof, selected from the groupconsisting of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40,Programmed Death-1 (PD-1), inducible T cell co-stimulator (ICOS),lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276 (B7-H3), LIGHT,(TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokinereceptor, integrins, Signaling Lymphocytic Activation Molecules (SLAMproteins), activating NK cell receptors, BTLA, a Toll ligand receptor,ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103,ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof.

An antigen binding molecule that specifically binds the anti-CD19 scFvFMC63 sequence (SEQ ID NO: 1) and optionally comprises a detectablelabel is provided. When it is decided to employ a detectable label, anydetectable label can be employed in the method, as described herein, andsuitable labels can be selected using a desired set of criteria.Examples of types of detectable labels include fluorescent labels (e.g.,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malachite green, stilbene, LuciferYellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640,Cy 5, Cy 5.5, LC Red 705, Oregon green, the Alexa-Fluor dyes (AlexaFluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, AlexaFluor 660, Alexa Fluor 680), Cascade Blue, Cas-cade Yellow andR-phycoerythrin (PE) (Molecular Probes), FITC, Rhodamine, and Texas Red(Pierce), Cy5, Cy5.5, Cy7 (Amersham Life Science)). Suitable opticaldyes, including fluorophores, are described in Johnson, Molecular ProbesHandbook: A Guide to Fluorescent Probes and Labeling Techniques, 11^(th)Edition, Life Technologies, (2010), hereby expressly incorporated byreference, radiolabels (e.g., isotope markers such as ³H, ¹¹C, ¹⁴C, ¹⁵N,¹⁸F, ³⁵S, ⁶⁴CU, ⁹⁹Y, ⁹⁹Tc, ¹¹¹In, ¹²⁴I, ¹²⁵I, ¹³¹I). Photochromiccompounds, a Halo-tag, Atto dyes, Tracy dyes, proteinaceous fluorescentlabels (e.g., proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., (1994) Science 263:802-805),EGFP (Clon-tech Labs., Inc., Genbank Accession Number U55762), bluefluorescent protein (BFP, Quantum Biotechnologies, Inc; Stauber, (1998)Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6: 178-182),enhanced yellow fluorescent protein (Clontech Labs., Inc.), luciferase(Ichiki et al., (1993) J. Immunol. 150:5408-5417), magnetic labels(e.g., DYNABEADS), etc can also be employed. Strategies for the labelingof proteins are well known in the art and can be employed in thedisclosed method. See, e.g., Obermaier et al., (2015) Methods Mol Biol1295:153-65; Strack (2016) Nature Methods 13:33; Site-Specific ProteinLabeling: Methods and Protocols, (Gautier and Hinner, eds.) 2015,Springer.

The label can be associated with the antigen binding molecule at anyposition in the molecule, although it is preferable to associate thelabel with the molecule at a position (or positions, if multiple labelsare employed) at a point such that the binding properties of themolecule are not modified (unless such modified binding activity isdesired). Any antigen binding molecule that specifically binds anti-CD19scFv FMC63 sequence (SEQ ID NO: 1) (or fragment thereof) can beemployed, such as those disclosed herein, e.g., those having one or moreof the CDRs shown in FIGS. 5-21.

The antigen binding molecule can be disposed on any surface, or nosurface at all. For example, the antigen binding molecule can be presentin a buffer and the buffer-antigen binding molecule can be contactedwith the sample. Alternatively, the antigen binding molecule can beassociated with a surface. Suitable surfaces include agarose beads,magnetic beads such as DYNABEADS, or a plastic, glass or ceramic platesuch as a welled plate, a bag such as a cell culture bag, etc. Thesurface can itself be disposed in another structure, such as a column.

Conditions that permit the formation of a binding complex will bedependent on a variety of factors, however generally aqueous buffers atphysiological pH and ionic strength, such as in phosphate-bufferedsaline (PBS), will favor formation of binding complexes and arepreferred in the disclosed method. Since the component parts of abinding complex can be disposed on surfaces as described herein, formedbinding complexes can also be disposed on surfaces.

At this stage, no binding complexes may have formed, or a plurality ofbinding complexes comprising one or more antigen binding molecules boundto a molecule comprising SEQ ID NO: 1 (or one or more moleculescomprising SEQ ID NO: 1 bound to an antigen binding molecule) may haveformed. Unbound molecules comprising SEQ ID NO: 1 and/or unbound antigenbinding molecules may also be present in the local environment of anyformed binding complexes.

Any molecules not part of a binding complex are then separated from anyformed binding complexes. The method of the removal will depend on thestructure and/or local environment of the binding complexes. Forexample, if the antigen binding molecule is disposed on a bead, plate orbag the unbound components of the reaction mixture can be washed awayusing a solution that leaves formed binding complexes intact. If abinding complex is disposed on a bead, the bead itself may be situatedin a column or other structure and the same approach can be used.

The solution used to induce the formation of binding complexes can beused, for example, as a wash solution to remove unbound components. Anysuitable buffer or solution that does not disrupt formed bindingcomplexes can also be used. Typically, buffers having high saltconcentrations, non-physiological pH, containing chaotropes ordenaturants, are preferably avoided when performing this step of themethod.

A formed binding complex is then separated into (a) a moleculecomprising SEQ ID NO: 1, and (b) an antigen binding molecule. Theseparation can be achieved using standard methodologies known to thoseof skill in the art. For example, a solution of suitable pH andcomposition can be washed over the complexes. A solution that iscommonly employed for this purpose is 0.1 M glycine HCl, pH 2.5-3.0, andthis solution can be employed to achieve the separation. Other solutionsthat can be employed include 100 mM citric acid, pH 3.0, 50-100 mMtriethylamine or triethanolamine, pH 11.5; 150 mM ammonium hydroxide, pH10.5; 0.1 M glycine.NaOH, pH 10.0; 5 M lithium chloride, 3.5 M magnesiumor potassium chloride, 3.0 M potassium chloride, 2.5 M sodium orpotassium iodide, 0.2-3.0 M sodium thiocyanate, 0.1 M Tris-acetate with2.0 M NaCl, pH 7.7; 2-6 M guanidine HCl, 2-8 M urea, 1.0 M ammoniumthiocyanate, 1% sodium deoxycholate 1% SDS; and 10% dioxane 50% ethyleneglycol, pH 8-11.5.

Following the separation, if the molecule comprising SEQ ID NO: 1 is ofprimary interest it can be collected; alternatively, if the antigenbinding molecule is of primary interest it can be collected.

Vd. Method of Determining the Presence or Absence of a Molecule

As disclosed herein, it may sometimes be desirable to isolate a moleculecomprising the anti-CD19 scFv FMC63 sequence (SEQ ID NO: 1), as providedherein. In other cases, simply knowing whether a molecule comprising SEQID NO: 1 provided herein is present or absent from a sample is enoughinformation. For example, it may be beneficial to know that such amolecule is being expressed, regardless of the level of expression. Inother cases, it may be desirable to know if a purification process orstep designed to remove such a molecule has been effectively performed.Thus, the qualitative determination of the presence or absence of amolecule comprising the anti-CD19 scFv FMC63 sequence (SEQ ID NO: 1) ofthe instant disclosure can be useful in multiple applications. In viewthereof, a method of determining the presence or absence of a moleculecomprising SEQ ID NO: 1 in a sample is provided.

In some embodiments, the method comprises providing a sample known orsuspected to comprise a molecule comprising SEQ ID NO: 1.

In some embodiments, the molecule comprising SEQ ID NO: 1 is a CAR. Whenthe molecule is a CAR it can comprise a molecule, or fragment thereof,selected from the group consisting of CD28, OX-40, 4-1BB/CD137, CD2,CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cellco-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1,CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276(B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gammareceptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulinprotein, cytokine receptor, integrins, Signaling Lymphocytic ActivationMolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE,CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof.

An antigen binding molecule comprising a detectable label thatspecifically binds the anti-CD19 scFv FMC63 (SEQ ID NO: 1) is provided.Suitable labels can be selected using a desired set of criteria.Examples of types of detectable labels include fluorescent labels (e.g.,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malachite green, stilbene, LuciferYellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640,Cy 5, Cy 5.5, LC Red 705, Oregon green, the Alexa-Fluor dyes (AlexaFluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, AlexaFluor 660, Alexa Fluor 680), Cascade Blue, Cas-cade Yellow andR-phycoerythrin (PE) (Molecular Probes), FITC, Rhodamine, and Texas Red(Pierce), Cy5, Cy5.5, Cy7 (Amersham Life Science). Suitable opticaldyes, including fluoro-phores, are described in Johnson, MolecularProbes Handbook: A Guide to Fluorescent Probes and Labeling Techniques,11^(th) Edition, Life Technologies, (2010), hereby expresslyincorporated by reference, radiolabels (e.g., isotope markers such as³H, ¹¹C, ¹⁴C, ¹⁵N, ¹⁸F, ³⁵S, ⁶⁴CU, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁴I, ¹²⁵I, ¹³¹I),photochromic compounds, a Halo-tag, Atto dyes, Tracy dyes, proteinaceousfluorescent labels (e.g., proteinaceous fluorescent labels also include,but are not limited to, green fluorescent protein, including a Renilla,Ptilosarcus, or Aequorea species of GFP (Chalfie et al., (1994) Science263:802-805), EGFP (Clon-tech Labs., Inc., Genbank Accession NumberU55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc;Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr.Biol. 6: 178-182), enhanced yellow fluorescent protein (Clontech Labs.,Inc.), luciferase (Ichiki et al., (1993) J. Immunol. 150:5408-5417),magnetic labels (e.g., DYNABEADS), etc. Strategies for the labeling ofproteins are well known in the art and can be employed in the disclosedmethod. See, e.g., Obermaier et al., (2015) Methods Mol Biol1295:153-65; Strack (2016) Nature Methods 13:33; Site-Specific ProteinLabeling: Methods and Protocols, (Gautier and Hinner, eds.) 2015,Springer.

The label can be associated with the antigen binding molecule at anyposition in the molecule, although it is preferable to associate thelabel with the molecule at a position (or positions, if multiple labelsare employed) at a point such that the binding properties of themolecule are not modified (unless such modified binding activity isdesired). Any antigen binding molecule that specifically binds theanti-CD19 scFv FMC63 sequence (SEQ ID NO: 1 or fragment thereof) can beemployed, such as those disclosed herein, e.g., those having one or moreof the CDRs shown in FIGS. 5-21.

Continuing, the sample is contacted with the antigen binding moleculeunder conditions that permit the formation of a binding complexcomprising a molecule comprising SEQ ID NO: 1 (which can be presented ona cell) present in the sample and the antigen binding molecule. Theantigen binding molecule can be disposed on any surface, or no surfaceat all. For example, the antigen binding molecule can be present in abuffer and the buffer-antigen binding molecule can be contacted with thesample. Alternatively, the antigen binding molecule can be associatedwith a surface. Suitable surfaces include agarose beads, magnetic beadssuch as DYNABEADS, or a plastic, glass or ceramic plate such as a welledplate, a bag such as a cell culture bag, etc. The surface can itself bedisposed in another structure, such as a column.

Conditions that permit the formation of a binding complex will bedependent on a variety of factors, however generally aqueous buffers atphysiological pH and ionic strength, such as in phosphate-bufferedsaline (PBS), will favor formation of binding complexes and arepreferred in the disclosed method. Since the component parts of abinding complex can be disposed on surfaces as described herein, formedbinding complexes can also be disposed on surfaces.

At this stage, no binding complexes may have formed, or a plurality ofbinding complexes comprising one or more antigen binding molecules boundto a molecule comprising the anti-CD19 scFv FMC63 sequence (SEQ IDNO: 1) (or one or more molecules comprising SEQ ID NO: 1 bound to anantigen binding molecule) may have formed. Unbound molecules comprisingSEQ ID NO: 1 and/or unbound antigen binding molecules may also bepresent in the local environment of any formed binding complexes.

Any molecules not part of a binding complex are then separated from anyformed binding complexes. The method of the removal will depend on thestructure and/or local environment of the binding complexes. Forexample, if the antigen binding molecule is disposed on a bead, plate orbag the unbound components of the reaction mixture can be washed awayusing a solution that leaves formed binding complexes intact. If abinding complex is disposed on a bead, the bead itself may be situatedin a column or other structure and the same approach can be used.

The solution used to induce the formation of binding complexes can beused, for example, as a wash solution to remove unbound components. Anysuitable buffer or solution that does not disrupt formed bindingcomplexes can also be used. Typically, buffers having high saltconcentrations, non-physiological pH, containing chaotropes ordenaturants, should be avoided when performing this step of the method.

Lastly, the presence or absence of a binding complex—which will comprisea molecule comprising SEQ ID NO: 1 and an antigen binding molecule—isdetected. The specific method employed to detect the presence or absenceof a binding complex will be dependent on the nature of the labelselected. For example, flow cytometry or FACS can be employed when afluorescent label is selected; when an isotope label is selected massspectrometry, NMR or other technique can be employed; magnetic-basedcell sorting can be employed when a magnetic label is chosen; microscopycan also be employed. The end result of the method is a qualitativeassessment of the presence or absence of the antigen binding moleculecomprising the detectable label, and thus, the presence or absence ofits binding partner, the molecule comprising SEQ ID NO: 1.

As is the case with all of the disclosed methods, the moleculecomprising the anti-CD19 scFv FMC63 sequence (SEQ ID NO: 1) can bedisposed in any environment. In preferred embodiments, the moleculecomprising SEQ ID NO: 1 is expressed on the surface of a cell. In thisembodiment, the cell can be of any type, and can be human or non-human(e.g., mouse, rate, rabbit, hamster, etc). In one preferred embodiment,the cell is an immune cell. An immune cell of the method can be any typeof immune cell (e.g., B lymphocytes, monocytes, dendritic cells,Langerhans cells, keratinocytes, endothelial cells, astrocytes,fibroblasts, and oligodendrocytes). T cells (including T cytotoxic, Thelper and Treg cells) are especially preferred. In specificembodiments, the cells are T cells, which can be obtained as describedherein and by methods known in the art. Any type of immune cell can beemployed in this embodiment of the disclosed method, and the cell can bea human or non-human cell (including both prokaryotic and eukaryoticcells). Exemplary cells include, but are not limited to immune cellssuch as T cells, tumor infiltrating lymphocytes (TILs), NK cells,TCR-expressing cells, dendritic cells, and NK-T cells. The T cells canbe autologous, allogeneic, or heterologous. In additional embodiments,the cells are T cells presenting a CAR. The T cells can be CD4+ T cellsor CD8+ T cells. When a T cell is employed in the disclosed methods, theT cell can be an in vivo T cell or an in vitro T cell.

In an additional embodiment, the cell can be disposed in, or isolatedfrom, any environment capable of maintaining the cell in a viable form,such as blood, tissue or any other sample obtained from a subject, cellculture media, tissue grown ex vivo, a suitable buffer, etc.

Ve. Method of Increasing the Concentration of a Molecule

Very often a molecule of interest is present in a sample inlower-than-desired levels. For example, when a cell is transfected witha foreign gene expression levels of the protein(s) encoded by theforeign gene are sometimes low. The same can be true for moleculessecreted from a cell; such molecules are often present in low quantities(but can still be detected using the methods provided herein, if themolecule comprises the anti-CD19 scFv FMC63 sequence (SEQ ID NO: 1). Onesolution to the problem of low expression levels is to increase theconcentration of the molecule of interest, which can be free insolution, or expressed on the surface of a cell. The concentration ofintracellularly-expressed molecules of interest can also be enhanced,however the cells must first be lysed to release the molecule. Toaddress this problem, a method of increasing the concentration of cellspresenting a molecule comprising the anti-CD19 scFv FMC63 sequence (SEQID NO: 1) is provided.

In some embodiments, the method comprises providing a sample comprisingcells known or suspected to present a molecule comprising the anti-CD19scFv FMC63 sequence (SEQ ID NO: 1).

In specific embodiments, the molecule comprising the sequence SEQ ID NO:1 is a CAR. When the molecule is a CAR it can comprise a molecule, orfragment thereof, selected from the group consisting of CD28, OX-40,4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1),inducible T cell co-stimulator (ICOS), lymphocyte function-associatedantigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3zeta, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a),DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins,an Immunoglobulin protein, cytokine receptor, integrins, SignalingLymphocytic Activation Molecules (SLAM proteins), activating NK cellreceptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1,GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma,IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX,CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand thatspecifically binds with CD83, and combinations thereof.

An antigen binding molecule that specifically binds SEQ ID NO: 1, andoptionally comprises a detectable label, is provided. When it is decidedto employ a detectable label, any detectable label can be employed inthe method, as described herein, and suitable labels can be selectedusing a desired set of criteria. Examples of types of detectable labelsinclude fluorescent labels (e.g., fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malachite green, stilbene, Lucifer Yellow, Cascade Blue, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680),Cascade Blue, Cas-cade Yellow and R-phycoerythrin (PE) (MolecularProbes), FITC, Rhodamine, and Texas Red (Pierce), Cy5, Cy5.5, Cy7(Amersham Life Science). Suitable optical dyes, including fluoro-phores,are described in Johnson, Molecular Probes Handbook: A Guide toFluorescent Probes and Labeling Techniques, 11^(th) Edition, LifeTechnologies, (2010), hereby expressly incorporated by reference,radiolabels (e.g., isotope markers such as ³H, ¹¹C, ¹⁴C, ¹⁵N, ¹⁸F, ³⁵S,⁶⁴CU, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁴I, ¹²⁵I, ¹³¹I), photochromic compounds, aHalo-tag, Atto dyes, Tracy dyes, proteinaceous fluorescent labels (e.g.,proteinaceous fluorescent labels also include, but are not limited to,green fluorescent protein, including a Renilla, Ptilosarcus, or Aequoreaspecies of GFP (Chalfie et al., (1994) Science 263:802-805), EGFP(Clon-tech Labs., Inc., Genbank Accession Number U55762), bluefluorescent protein (BFP, Quantum Biotechnologies, Inc; Stauber, (1998)Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6: 178-182),enhanced yellow fluorescent protein (Clontech Labs., Inc.), luciferase(Ichiki et al., (1993) J. Immunol. 150:5408-5417), magnetic labels(e.g., DYNABEADS), etc. Strategies for the labeling of proteins are wellknown in the art and can be employed in the disclosed method. See, e.g.,Obermaier et al., (2015) Methods Mol Biol 1295:153-65; Strack (2016)Nature Methods 13:33; Site-Specific Protein Labeling: Methods andProtocols, (Gautier and Hinner, eds.) 2015, Springer.

The label can be associated with the antigen binding molecule at anyposition in the molecule, although it is preferable to associate thelabel with the molecule at a position (or positions, if multiple labelsare employed) at a point such that the binding properties of themolecule are not modified (unless such modified binding activity isdesired). Any antigen binding molecule that specifically binds theanti-CD19 scFv FMC63 sequence (SEQ ID NO: 1) (or fragment thereof) canbe employed, such as those disclosed herein, e.g., those having one ormore of the CDRs shown in FIGS. 5-21.

The antigen binding molecule can be disposed on any surface, or nosurface at all. For example, the antigen binding molecule can be presentin a buffer and the buffer-antigen binding molecule can be contactedwith the sample. Alternatively, the antigen binding molecule can beassociated with a surface. Suitable surfaces include agarose beads,magnetic beads such as DYNABEADS, or a plastic, glass or ceramic platesuch as a welled plate, a bag such as a cell culture bag, etc. Thesurface can itself be disposed in another structure, such as a column.

A cell presenting a molecule comprising SEQ ID NO: 1 can be of any type,and can be human or non-human (e.g., mouse, rate, rabbit, hamster, etc).In a preferred embodiment, the cell is an immune cell. An immune cell ofthe method can be any type of immune cell (e.g., B lymphocytes,monocytes, dendritic cells, Langerhans cells, keratinocytes, endothelialcells, astrocytes, fibroblasts, and oligodendrocytes). T cells(including T cytotoxic, T helper and Treg cells) are especiallypreferred. In specific embodiments, the cells are T cells, which can beobtained as described herein and by methods known in the art. Any typeof immune cell can be employed in this embodiment of the disclosedmethod, and the cell can be a human or non-human cell (including bothprokaryotic and eukaryotic cells). Exemplary cells include, but are notlimited to immune cells such as T cells, tumor infiltrating lymphocytes(TILs), NK cells, TCR-expressing cells, dendritic cells, and NK-T cells.The T cells can be autologous, allogeneic, or heterologous. Inadditional embodiments, the cells are T cells presenting a CAR. The Tcells can be CD4+ T cells or CD8+ T cells. When a T cell is employed inthe disclosed methods, the T cell can be an in vivo T cell or an invitro T cell. Moreover, the cells can be disposed in, or isolated from,any environment capable of maintaining the cells in a viable form, suchas blood, tissue or any other sample obtained from a subject, cellculture media, tissue grown ex vivo, a suitable buffer, etc.

The sample comprising cells is contacted with the antigen bindingmolecule, under conditions that permit the formation of a bindingcomplex comprising a molecule comprising anti-CD19 scFv FMC63 sequence(SEQ ID NO: 1) and the antigen binding molecule. Conditions that permitthe formation of a binding complex will be dependent on a variety offactors, however generally aqueous buffers at physiological pH and ionicstrength, such as in phosphate-buffered saline (PBS), will favorformation of binding complexes and are preferred in the disclosedmethod. Since the component parts of a binding complex can be disposedon surfaces as described herein, formed binding complexes can also bedisposed on surfaces.

At this stage, no binding complexes may have formed, or a plurality ofbinding complexes comprising one or more antigen binding molecules boundto a molecule comprising the anti-CD19 scFv FMC63 sequence (SEQ IDNO: 1) (or one or more molecules comprising SEQ ID NO: 1 bound to anantigen binding molecule) may have formed. Unbound molecules comprisingSEQ ID NO: 1 and/or unbound antigen binding molecules may also bepresent in the local environment of any formed binding complexes.

Any molecules or cells not part of a binding complex are then separatedfrom any formed binding complexes. The method of the removal will dependon the structure and/or local environment of the binding complexes. Forexample, if the antigen binding molecule is disposed on a bead, plate orbag the unbound components of the reaction mixture can be washed awayusing a solution that leaves formed binding complexes intact. If abinding complex is disposed on a bead, the bead itself may be situatedin a column or other structure and the same approach can be used.

The solution used to induce the formation of binding complexes can beused, for example, as a wash solution to remove unbound components. Anysuitable buffer or solution that does not disrupt formed bindingcomplexes can also be used. Typically, buffers having high saltconcentrations, non-physiological pH, containing chaotropes ordenaturants, should be avoided when performing this step of the method.

At this stage of the method, a population of cells presenting a moleculecomprising the SEQ ID NO: 1 will be present. If a detectable label wasemployed, the concentration of the cells can be easily determined,consistent with the nature of the label. Cells not expressing themolecule comprising SEQ ID NO: 1 will be absent, and thus the population(or concentration) of cells presenting a molecule comprising SEQ ID NO:1 will be increased compared to the levels prior to performing themethod.

If the concentration of the molecule comprising the anti-CD19 scFv FMC63sequence (SEQ ID NO: 1) is not at a desired level, the above steps canbe repeated a desired number of times. In the context of this step ofthe method, a desired number of times can also be zero, if the desiredconcentration of cells is already present.

Vf. Method of Depleting a Population of Immune Cells

When a subject has an immune cell-mediated condition, it can be ofsignificant importance that the condition be controlled in a timelyfashion so as to prevent harm to the subject. For example, when asubject has an autoimmune reaction it may be desirable to suppress animmune cell-mediated response by depleting a population of immune cells,in an effort to prevent harm. In another example, a subject receivingimmunotherapy may react too strongly to the therapy and be at risk ofharm; depleting the population of immune cells administered to thesubject may be an effective approach to mitigating the subject'sreaction to the immunotherapy. In view of the need for a method ofcontrolling a subject's immune cell-mediated response, a method ofdepleting a population of immune cells presenting a molecule comprisingthe anti-CD19 scFv FMC63 sequence (SEQ ID NO: 1) is provided. An antigenbinding molecule that specifically recognizes SEQ ID NO: 1, e.g., thosehaving one or more of the CDRs shown in FIG. 6, can be employed in themethod.

In some embodiments, the method comprises providing a population ofimmune cells to be depleted, wherein the cells are known or suspected tobe expressing a molecule comprising the anti-CD19 scFv FMC63 sequence(SEQ ID NO: 1).

In specific embodiments, the molecule comprising SEQ ID NO: 1 is a CAR.When the molecule is a CAR it can comprise a molecule, or fragmentthereof, selected from the group consisting of CD28, OX-40, 4-1BB/CD137,CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cellco-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1,CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD247, CD276(B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gammareceptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulinprotein, cytokine receptor, integrins, Signaling Lymphocytic ActivationMolecules (SLAM proteins), activating NK cell receptors, BTLA, a Tollligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4,CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE,CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, andcombinations thereof.

An immune cell expressing a molecule comprising the anti-CD19 scFv FMC63(SEQ ID NO: 1) sequence can be of any type, and can be human ornon-human (e.g., mouse, rate, rabbit, hamster, etc). An immune cell ofthe method can be any type of immune cell (e.g., B lymphocytes,monocytes, dendritic cells, Langerhans cells, keratinocytes, endothelialcells, astrocytes, fibroblasts, and oligodendrocytes). T cells(including T cytotoxic, T helper and Treg cells) are especiallypreferred. In specific embodiments, the cells are T cells, which can beobtained as described herein and by methods known in the art. Any typeof immune cell can be employed in this embodiment of the disclosedmethod, and the cell can be a human or non-human cell (including bothprokaryotic and eukaryotic cells). Exemplary cells include, but are notlimited to immune cells such as T cells, tumor infiltrating lymphocytes(TILs), NK cells, TCR-expressing cells, dendritic cells, and NK-T cells.The T cells can be autologous, allogeneic, or heterologous. Inadditional embodiments, the cells are T cells presenting a CAR. The Tcells can be CD4+ T cells or CD8+ T cells. When a T cell is employed inthe disclosed methods, the T cell can be an in vivo T cell or an invitro T cell. Moreover, the cells can be disposed in, or isolated from,any environment capable of maintaining the cells in a viable form, suchas blood, tissue or any other sample obtained from a subject, cellculture media, tissue grown ex vivo, a suitable buffer, etc. As thedisclosed method can be employed in therapeutic settings, in preferredembodiments the population of immune cells are disposed in a subject,and more preferably a human subject.

Continuing, the immune cells are contacted with an antigen bindingmolecule that specifically binds to (a) the molecule comprising SEQ IDNO: 1, and (b) an activating molecule expressed on the surface of theimmune cell not expressing the molecule comprising SEQ ID NO: 1, underconditions that permit the formation of a ternary binding complexcomprising the molecule comprising SEQ ID NO: 1, the activating moleculeand the antigen binding molecule. The antigen binding molecule can bedisposed on any surface, or no surface at all. For example, the antigenbinding molecule (which can also comprise the population of immune cellsto be depleted and/or can be present in a buffer) and the buffer-antigenbinding molecule can be contacted with the sample. Alternatively, theantigen binding molecule can be associated with a surface. Suitablesurfaces include agarose beads, magnetic beads such as DYNABEADS, or aplastic, glass or ceramic plate such as a welled plate, a bag such as acell culture bag, etc. The surface can itself be disposed in anotherstructure, such as a column.

The immune cells are contacted with the antigen binding molecule, underconditions that permit the formation of a ternary binding complexcomprising a molecule comprising the anti-CD19 scFv FMC63 (SEQ ID NO: 1)sequence, the antigen binding molecule and an activating moleculeexpressed on the surface of an immune cell not expressing the moleculecomprising SEQ ID NO: 1. Conditions that permit the formation of abinding complex will be dependent on a variety of factors, howevergenerally aqueous buffers at physiological pH and ionic strength, suchas in phosphate-buffered saline (PBS), will favor formation of bindingcomplexes and are preferred in the disclosed method. Since the componentparts of a binding complex can be disposed on surfaces as describedherein, formed binding complexes can also be disposed on surfaces.

In preferred embodiments, the contacting is performed by administeringthe antigen binding molecule directly to a subject. In this embodiment,the subject will already have a population of cells to be depleted,wherein the cells express a molecule comprising the anti-CD19 scFv FMC63(SEQ ID NO: 1) sequence. Thus, these cells, as well as cells presentingan activating molecule, will be present in the subject prior to theadministration of the antigen binding molecule to the subject. The humanblood, lymph and tissue environment will permit the formation of ternarybinding complexes. The binding of the antigen binding molecule with themolecule comprising the anti-CD19 scFv FMC63 (SEQ ID NO: 1) sequenceserves to “tag” those cells presenting the molecule comprising SEQ IDNO: 1 (i.e., the cells to be depleted). This binding event may or maynot lead to depletion on its own. When the antigen binding moleculebinds the activating molecule to form the ternary binding complex,however, this binding event brings both cells (i.e., the cell expressingthe molecule comprising SEQ ID NO: 1, and the cell expressing theactivating molecule) together into proximity. The physiological resultof the binding event is the killing of the cell expressing the moleculecomprising SEQ ID NO: 1. Thus, with multiple binding events occurringthroughout the subject the population of immune cells bearing themolecule comprising SEQ ID NO: 1 are depleted and the risk of harm tothe subject decreases.

Sequences and SEQ ID Nos

The instant disclosure comprises a number of nucleic acid andpolypeptide sequences. For convenience, the table below correlates eachsequence with its appropriate description and SEQ ID NO.

SEQ ID NO Description Sequence  1 Anti-CD19 scFvDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP FMC63DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGT SVTVSS  2 Clone 7 VHATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGGAGCAGCTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGAGGAACCCTGACAGTCACCTGCAAAGCCTCTGGATTCTCCTTCAGTAACAATGGAATTTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGTCTTTATGTTGGTAGTAGTGATACCACTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAAGCTCGTCGACCACGGTGACTCTACAAATGACCAGTCTGACAGTCGCGGACACGGCCACCTATTTCTGTACGATAAATCTCGGCTTGTGGGGCCCCGGCACCC TGGTCACCGTCTCCTCA  3 Clone 7 VHMETGLRWLLLVAVLKGVQCQEQLEESGGDLVKPGGTLTVTCKASGFSFSNNGICWVRQAPGKGLEWIGCLYVGSSDTTYYASWAKGRFTISKSSSTTVTLQMTSLTVADTATYFCTI NLGLWGPGTLVTVSS  4 Clone 7 HCMETGLRWLLLVAVLKGVQCQEQLEESGGDLVKPGGTLTVTCKASGFSFSNNGICWVRQAPGKGLEWIGCLYVGSSDTTYYASWAKGRFTISKSSSTTVTLQMTSLTVADTATYFCTINLGLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMH EALHNHYTQKSISRSPGK  5Clone 7 VH CDR1 GFSFSNN  6 Clone 7 VH CDR2 YVGSSD  7 Clone 7 VH CDR3NLGL  8 Clone 7 VL ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCATCGTGGTGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGAGTGTTTATAATAGCGACTGGTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATGCTGCATCCACTCTGGCATCTGGGGTCCCATCGCGCTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGATGTGGTGTGTGACGATGCTGCCACTTATTATTGTGCAGGATATAAAAGTAGTAGTACTGATGGGATTGCTTTCGGCGGA GGGACCGAGGTGGTGGTCAAA  9Clone 7 VL MDTRAPTQLLGLLLLWLPGATFAIVVTQTPSSKSVPVGGTVTINCQASESVYNSDWLAWYQQKPGQPPKQLIYAASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSSS TDGIAFGGGTEVVVK 10 Clone 7 LCMDTRAPTQLLGLLLLWLPGATFAIVVTQTPSSKSVPVGGTVTINCQASESVYNSDWLAWYQQKPGQPPKQLIYAASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSSSTDGIAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC 11 Clone 7 VL CDR1QASESVYNSDWLA 12 Clone 7 VL CDR2 AASTLAS 13 Clone 7 VL CDR3 AGYKSSSTDGIA14 Clone 13 VH ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGGAGCAGCTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGAGGAACCCTGACAGTCACCTGCAAAGCCTCTGGATTCTCCTTCAGTAACAATGGAATTTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGTCTTTATGTTGGTAGTAGTGATACCACTTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAAGCTCGTCGACCACGGTGACTCTACAAATGACCAGTCTGACAGTCGCGGACACGGCCACCTATTTCTGTACGATAAATCTCGGCTTGTGGGGCCCCGGCACCC TGGTCACCGTCTCCTCA 15 Clone 13 VHMETGLRWLLLVAVLKGVQCQEQLEESGGDLVKPGGTLTVTCKASGFSFSNNGICWVRQAPGKGLEWIGCLYVGSSDTTYYASWAKGRFTISKSSSTTVTLQMTSLTVADTATYFCTI NLGLWGPGTLVTVSS 16 Clone 13 HCMETGLRWLLLVAVLKGVQCQEQLEESGGDLVKPGGTLTVTCKASGFSFSNNGICWVRQAPGKGLEWIGCLYVGSSDTTYYASWAKGRFTISKSSSTTVTLQMTSLTVADTATYFCTINLGLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMH EALHNHYTQKSISRSPGK  5Clone 13 VH CDR1 GFSFSNN  6 Clone 13 VH CDR2 YVGSSD  7 Clone 13 VH CDR3NLGL 17 Clone 13 VL ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACACTTGCCATCGTGGTGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGAGTGTTTATAATAGCGACTGGTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATGCTGCATCCACTCTGGCATCTGGGGTCCCATCGCGCTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGATGTGGTGTGTGACGATGCTGCCACTTATTATTGTGCAGGATATAAAAGTAGTAGTACTGATGGGATTGCTTTCGGCGGA GGGACCGAGGTGGTGGTCAAA 18Clone 13 VL MDTRAPTQLLGLLLLWLPGATLAIVVTQTPSSKSVPVGGTVTINCQASESVYNSDWLAWYQQKPGQPPKQLIYAASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSSS TDGIAFGGGTEVVVK 19 Clone 13 LCMDTRAPTQLLGLLLLWLPGATLAIVVTQTPSSKSVPVGGTVTINCQASESVYNSDWLAWYQQKPGQPPKQLIYAASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSSSTDGIAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC 11 Clone 13 VL CDR1QASESVYNSDWLA 12 Clone 13 VL CDR2 AASTLAS 13 Clone 13 VL CDR3AGYKSSSTDGIA 20 Clone 14-1 VH ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGGAGCAGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCAAAGCCTCTGGATTCGACTTCAGTATCAACTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGTTGGAGTGGATCGCATGCATTTATACTGGTGATGATGACACTTTCTACGCGAGCTGGGCGAAAGGCCGGTTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTACAACTGAACAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGTGAGAGGTCTATATAGTGGTAGTATTAATAACCTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA 21 Clone 14-1VHMETGLRWLLLVAVLKGVQCQEQLEESGGGLVKPGASLTLTCKASGFDFSINYYMCWVRQAPGKGLEWIACIYTGDDDTFYASWAKGRFTISKTSSTTVTLQLNSLTAADTATYFCVRG LYSGSINNLWGPGTLVTVSS 22Clone 14-1 HC METGLRWLLLVAVLKGVQCQEQLEESGGGLVKPGASLTLTCKASGFDFSINYYMCWVRQAPGKGLEWIACIYTGDDDTFYASWAKGRFTISKTSSTTVTLQLNSLTAADTATYFCVRGLYSGSINNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK 23Clone 14-1VH GFDFSINY CDR1 24 Clone 14-1VH YTGDD CDR2 25 Clone 14-1VHGLYSGSINNL CDR3 26 Clone 14-1VL ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGATGCCAGATGTGCGCTTGTGATGACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGGCCAGTCAGAGTGTTTATAACAACGACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAACTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCGCTTACTATTGTGCAGGCGTTAAAGGTTATAGTAATGATAATAATGGTTTCGGCGG AGGGACCGAGGTGGTGGTCAAA 27Clone 14-1VL MDTRAPTQLLGLLLLWLPDARCALVMTQTPSPVSAAVGGTVTISCQASQSVYNNDYLSWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAAAYYCAGVKGY SNDNNGFGGGTEVVVK 28 Clone 14-1LCMDTRAPTQLLGLLLLWLPDARCALVMTQTPSPVSAAVGGTVTISCQASQSVYNNDYLSWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAAAYYCAGVKGYSNDNNGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGD C 29 Clone 14-1 VL QASQSVYNNDYLS CDR1 30 Clone 14-1 VL YASTLAS CDR2 31 Clone 14-1 VLAGVKGYSNDNNG CDR3 32 Clone 14-7 VHATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAATGTCAGTCGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCAAAGCCTCTGGATTCGACTTCAGTATCAACTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGTTGGAGTGGATCGCATGCATTTATACTGGTGATGATGACACTTTCTACGCGAGCTGGGCGAAAGGCCGGTTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTACAACTGAACAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGTGAGAGGTCTATATAGTGGTAGTATTAATAACCTGTG GGGCCCAGGCACCCTGGTCACCGTCTCCTCA33 Clone 14-7 VH METGLRWLLLVAVLKGVQCQSLEESGGGLVKPGASLTLTCKASGFDFSINYYMCWVRQAPGKGLEWIACIYTGDDDTFYASWAKGRFTISKTSSTTVTLQLNSLTAADTATYFCVRGL YSGSINNLWGPGTLVTVSS 34Clone 14-7 HC METGLRWLLLVAVLKGVQCQSLEESGGGLVKPGASLTLTCKASGFDFSINYYMCWVRQAPGKGLEWIACIYTGDDDTFYASWAKGRFTISKTSSTTVTLQLNSLTAADTATYFCVRGLYSGSINNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSV MHEALHNHYTQKSISRSPGK 23Clone 14-7 VH GFDFSINY CDR1 24 Clone 14-7 VH YTGDD CDR2 25 Clone 14-7 VHGLYSGSINNL CDR3 35 Clone 14-7 VL ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGATGCCAGATGTGCGCTTGTGATGACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGGCCAGTCAGAGTGTTTATAACAACGACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAACTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCGCTTACTATTGTGCAGGCGTTAAAGGTTATAGTAATGATAATAATGGTTTCGGCGG AGGGACCGAGGTGGTGGTCAAA 36Clone 14-7 VL MDTRAPTQLLGLLLLWLPDARCALVMTQTPSPVSAAVGGTVTISCQASQSVYNNDYLSWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAAAYYCAGVKGY SNDNNGFGGGTEVVVK 37Clone 14-7 LC MDTRAPTQLLGLLLLWLPDARCALVMTQTPSPVSAAVGGTVTISCQASQSVYNNDYLSWYQQKPGQPPKLLIYYASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAAAYYCAGVKGYSNDNNGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGD C 29 Clone 14-7 VLQASQSVYNNDYLS CDR1 30 Clone 14-7 VL YASTLAS CDR2 31 Clone 14-7 VLAGVKGYSNDNNG CDR3 38 Clone 15 VH ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGGGTCCAGTGTCAGTCGTTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCTTCACGAGCAACTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGCGTGCATTTTTCTTGGTAGTAGTGGTAACACTGTCTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGTCGCGGACACGGCCACCTATTTCTGTGCGAGAGACTATGTTAATGGTTATGACTACTTTAACTTGTGGGGCCCAGGCACCTTGGTCACCGTCTCCTCA 39 Clone 15 VHMETGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCTASGFSFTSNYYMCWVRQAPGKGLEWVACIFLGSSGNTVYANWAKGRFTISKTSSTTVTLQMTSLTVADTATYFCAR DYVNGYDYFNLWGPGTLVTVSS 40Clone 15 HC METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCTASGFSFTSNYYMCWVRQAPGKGLEWVACIFLGSSGNTVYANWAKGRFTISKTSSTTVTLQMTSLTVADTATYFCARDYVNGYDYFNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVF TCSVMHEALHNHYTQKSISRSPGK 41Clone 15 VH CDR1 GFSFTSNY 42 Clone 15 VH CDR2 FLGSSG 43 Clone 15 VH CDR3DYVNGYDYFNL 44 Clone 15 VL ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCGGCTGTTGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATAATAAGAACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAAGGCCTGATCTATTCTACATCGACTCTAGATTCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCAGTTATGATTGTAGTAGTGCTGATTGTAATGCTTTCGGCGGAGG GACCGAGGTGGTGGTCAAA 45Clone 15 VL MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAAVGGTVTINCQSSQSVYNKNLAWYQQKPGQPPKGLIYSTSTLDSGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSS ADCNAFGGGTEVVVK 46 Clone 15 LCMDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAAVGGTVTINCQSSQSVYNKNLAWYQQKPGQPPKGLIYSTSTLDSGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSSADCNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC 47 Clone 15 VL CDR1QSSQSVYNKNLA 48 Clone 15 VL CDR2 STSTLDS 49 Clone 15 VL CDR3LGSYDCSSADCNA 50 Clone 17 VH ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAATGTCAGTCGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCTTCAGTGACAGTTGGTACTTGTGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTTATACTGGTGATGGTGACACTTATTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAGACCTCGTCGACCACAGTGACTCTACAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGCGAGGGGTGCCCAATTTTACTTGTGGGGCCAAGGCACC CTGGTCACCGTCTCCTCA 51 Clone 17 VHMETGLRWLLLVAVLKGVQCQSLEESGGGLVKPGASLTLTCTASGFSFSDSWYLCWVRQAPGKGLEWIACIYTGDGDTYYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARG AQFYLWGQGTLVTVSS 52 Clone 17 HCMETGLRWLLLVAVLKGVQCQSLEESGGGLVKPGASLTLTCTASGFSFSDSWYLCWVRQAPGKGLEWIACIYTGDGDTYYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGAQFYLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMH EALHNHYTQKSISRSPGK 53Clone 17 VH CDR1 GFSFSDSW 54 Clone 17 VH CDR2 YTGDG 55 Clone 17 VH CDR3GAQFYL 56 Clone 17 VL ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAGGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATGCCAACACCTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTGCATCCAGTCTGGCATCTGGGGTCCCACCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCGCTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCAGATATAGTTGTGGTCTTGCTGATTGTGCTGCTTTCGGCG GAGGGACCGAGGTGGTGGTCAAA 57Clone 17 VL MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAAVGGTVTINCQSSQSVYANTYLSWYQQKPGQPPKQLIYSASSLASGVPPRFKGSGSGTQFALTISDVQCDDAATYYCLGRYSCG LADCAAFGGGTEVVVK 58 Clone 17 LCMDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAAVGGTVTINCQSSQSVYANTYLSWYQQKPGQPPKQLIYSASSLASGVPPRFKGSGSGTQFALTISDVQCDDAATYYCLGRYSCGLADCAAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGD C 59 Clone 17 VL CDR1QSSQSVYANTYLS 60 Clone 17 VL CDR2 SASSLAS 61 Clone 17 VL CDR3LGRYSCGLADCAA 11 7 VL CDR1 Kabat QASESVYNSDWLA 12 7 VL CDR2 KabatAASTLAS 13 7 VL CDR3 Kabat AGYKSSSTDGIA 11 13 VL CDR1 KabatQASESVYNSDWLA 12 13 VL CDR2 Kabat AASTLAS 13 13 VL CDR3 KabatAGYKSSSTDGIA 29 14-1 VL CDR1 QASQSVYNNDYLS Kabat 30 14-1 VL CDR2 YASTLASKabat 31 14-1 VL CDR3 AGVKGYSNDNNG Kabat 29 14-7 VL CDR1 QASQSVYNNDYLSKabat 30 14-7 VL CDR2 YASTLAS Kabat 31 14-7 VL CDR3 AGVKGYSNDNNG Kabat47 15 VL CDR1 Kabat QSSQSVYNKNLA 48 15 VL CDR2 Kabat STSTLDS 4915 VL CDR3 Kabat LGSYDCSSADCNA 59 17 VL CDR1 Kabat QSSQSVYANTYLS 6017 VL CDR2 Kabat SASSLAS 61 17 VL CDR3 Kabat LGRYSCGLADCAA 627 VH CDR1 Kabat NNGIC 63 7 VH CDR2 Kabat CLYVGSSDTTYYASWAK  77 VH CDR3 Kabat NLGL 62 13 VH CDR1 Kabat NNGIC 63 13 VH CDR2 KabatCLYVGSSDTTYYASWAK  7 13 VH CDR3 Kabat NLGL 64 14-1 VH CDR1 INYYMC Kabat65 14-1 VH CDR2 CIYTGDDDTFYASWAK Kabat 25 14-1 VH CDR3 GLYSGSINNL Kabat64 14-7 VH CDR1 INYYMC Kabat 65 14-7 VH CDR2 CIYTGDDDTFYASWAK Kabat 2514-7 VH CDR3 GLYSGSINNL Kabat 66 15 VH CDR1 Kabat SNYYMC 6715 VH CDR2 Kabat CIFLGSSGNTVYANWAK 43 15 VH CDR3 Kabat DYVNGYDYFNL 6817 VH CDR1 Kabat DSWYLC 69 17 VH CDR2 Kabat CIYTGDGDTYYATWAK 5517 VH CDR3 Kabat GAQFYL 11 7 VL CDR1 Chothia QASESVYNSDWLA 127 VL CDR2 Chothia AASTLAS 13 7 VL CDR3 Chothia AGYKSSSTDGIA 1113 VL CDR1 QASESVYNSDWLA Chothia 12 13 VL CDR2 AASTLAS Chothia 1313 VL CDR3 AGYKSSSTDGIA Chothia 29 14-1 VL CDR1 QASQSVYNNDYLS Chothia 3014-1 VL CDR2 YASTLAS Chothia 31 14-1 VL CDR3 AGVKGYSNDNNG Chothia 2914-7 VL CDR1 QASQSVYNNDYLS Chothia 30 14-7 VL CDR2 YASTLAS Chothia 3114-7 VL CDR3 AGVKGYSNDNNG Chothia 47 15 VL CDR1 QSSQSVYNKNLA Chothia 4815 VL CDR2 STSTLDS Chothia 49 15 VL CDR3 LGSYDCSSADCNA Chothia 5917 VL CDR1 QSSQSVYANTYLS Chothia 60 17 VL CDR2 SASSLAS Chothia 6117 VL CDR3 LGRYSCGLADCAA Chothia  5 7 VH CDR1 GFSFSNN Chothia  67 VH CDR2 YVGSSD Chothia  7 7 VH CDR3 NLGL Chothia  5 13 VH CDR1 GFSFSNNChothia  6 13 VH CDR2 YVGSSD Chothia  7 13 VH CDR3 NLGL Chothia 2314-1 VH CDR1 GFDFSINY Chothia 24 14-1 VH CDR2 YTGDD Chothia 2514-1 VH CDR3 GLYSGSINNL Chothia 23 14-7 VH CDR1 GFDFSINY Chothia 2414-7 VH CDR2 YTGDD Chothia 25 14-7 VH CDR3 GLYSGSINNL Chothia 4115 VH CDR1 GFSFTSNY Chothia 42 15 VH CDR2 FLGSSG Chothia 43 15 VH CDR3DYVNGYDYFNL Chothia 53 17 VH CDR1 GFSFSDSW Chothia 54 17 VH CDR2 YTGDGChothia 55 17 VH CDR3 GAQFYL Chothia 11 7 VL CDR1 IMGT QASESVYNSDWLA 127 VL CDR2 IMGT AASTLAS 13 7 VL CDR3 IMGT AGYKSSSTDGIA 11 13 VL CDR1 IMGTQASESVYNSDWLA 12 13 VL CDR2 IMGT AASTLAS 13 13 VL CDR3 IMGT AGYKSSSTDGIA29 14-1 VL CDR1 QASQSVYNNDYLS IMGT 30 14-1 VL CDR2 YASTLAS IMGT 3114-1 VL CDR3 AGVKGYSNDNNG IMGT 29 14-7 VL CDR1 QASQSVYNNDYLS IMGT 3014-7 VL CDR2 YASTLAS IMGT 31 14-7 VL CDR3 AGVKGYSNDNNG IMGT 4715 VL CDR1 IMGT QSSQSVYNKNLA 48 15 VL CDR2 IMGT STSTLDS 4915 VL CDR3 IMGT LGSYDCSSADCNA 59 17 VL CDR1 IMGT QSSQSVYANTYLS 6017 VL CDR2 IMGT SASSLAS 61 17 VL CDR3 IMGT LGRYSCGLADCAA 707 VH CDR 1 IMGT GFSFSNNGIC 63 7 VH CDR 2 IMGT CLYVGSSDTTYYASWAK  77 VH CDR 3 IMGT NLGL 70 13 VH CDR 1 GFSFSNNGIC IMGT 63 13 VH CDR 2CLYVGSSDTTYYASWAK IMGT  7 13 VH CDR 3 NLGL IMGT 71 14-1 VH CDR 1GFDFSINYYMC IMGT 65 14-1 VH CDR 2 CIYTGDDDTFYASWAK IMGT 25 14-1 VH CDR 3GLYSGSINNL IMGT 71 14-7 VH CDR 1 GFDFSINYYMC IMGT 65 14-7 VH CDR 2CIYTGDDDTFYASWAK IMGT 25 14-7 VH CDR 3 GLYSGSINNL IMGT 72 15 VH CDR 1GFSFTSNYYMC IMGT 67 15 VH CDR 2 CIFLGSSGNTVYANWAK IMGT 43 15 VH CDR 3DYVNGYDYFNL IMGT 73 17 VH CDR 1 GFSFSDSWYLC IMGT 69 17 VH CDR 2CIYTGDGDTYYATWAK IMGT 55 17 VH CDR 3 GAQFYL IMGT 74 HUMANIZEDRASQDISKYLN FMC 63 VL CDR1 75 HUMANIZED HTSRLHS FMC 63 VL CDR2 76HUMANIZED QQGNTLPYT FMC 63 VL CDR3 77 HUMANIZED GVSLPDY FMC 63 VH CDR1Clothia 78 HUMANIZED WGSET FMC 63 VH CDR2 Clothia 79 HUMANIZEDHYYYGGSYAMDY FMC 63 VH CDR3 80 HUMANIZED DYGVS FMC 63 VH CDR1 Kabat 81HUMANIZED VIWGSETTYYNSALKS FMC 63 VH CDR2 Kabat/IMGT 82 HUMANIZEDGVSLPDYGVS FMC 63 VH CDR1 IMGT

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.However, the citation of a reference herein should not be construed asan acknowledgement that such reference is prior art to the presentinvention. To the extent that any of the definitions or terms providedin the references incorporated by reference differ from the terms anddiscussion provided herein, the present terms and definitions control.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and Examples that follow detail certain preferredembodiments of the invention and describe the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

EXAMPLES

The present invention is further illustrated by the following examples,which should not be construed as further limiting. The contents of allreferences cited throughout this application are expressly incorporatedherein by reference.

Example 1 Generation and Screening of Antigen Binding Molecules

Monoclonal antibodies were generated through immunization of rabbitsusing the anti-CD19 scFv FMC63 (SEQ ID NO: 1), conjugated to Fc asimmunogen. Titer was determined via screening polyclonal sera by usingFc as a screen. A secondary screen was performed using CAR T cellsassayed via flow cytometry. Once titer was achieved, the immunizedrabbits were sacrificed and monoclonals were derived using standardhybridoma generation and subcloning techniques. The final screening ofthe hybridoma subclones was accomplished via additional rounds of flowcytometry and immunohistochemistry (IHC) of proliferating CAR T cells orfixed cell pellets derived from CAR T cells, respectively. The sequencesof the final two subclones selected were determined by standard Sangersequencing of the hybridomas subclones.

PBMCs were isolated from healthy donor leukopaks (Hemacare™) usingFicoll-Paque density centrifugation per manufacturer's instructions.PBMCs were stimulated using OKT3 (50 ng/ml, Miltenyi Biotec™) in R10media+IL-2 (300 IU/ml, Proleukin®, Prometheus® Therapeutics andDiagnostics). Two days after stimulation, CAR T cells presenting theanti-CD19 scFv FMC63 (SEQ ID NO: 1) were generated through viraltransduction of these activated primary human T cells. Transduction wasperformed using either a retro- (pMSVG vector) or lentivirus (pGARvector) depending upon the origin of the CARs used in the screening.Confirmation of CAR construct expression and viral transductionefficiency was determined using Protein L conjugated to phycoerythrin(PE) or fluorescein isothiocyanate (FITC). Results are shown in FIGS.1A-1B and 2.

Example 2 Immunohistochemistry (IHC) Studies

Several antibodies disclosed herein, (Clones 7 and 13) were assessedwith respect to their ability to function as reagents inimmunohistochemistry (IHC) studies. To create the fixed cell pellets forIHC staining, ˜2e6 CAR T cells presenting the anti-CD19 scFv FMC63 (SEQID NO: 1) were centrifuged and washed with PBS. The cells wereresuspended in PBS containing 0.45% paraformaldehyde (PFA) and incubatedon a shaking platform for 2 hours at room temperature. After theincubation, the cells were washed once more with PBS and resuspended inPBS with 5% BSA. The results of the IHC experiments are shown in FIGS. 3and 4A (FIG. 4B shows controls), and demonstrate that at least clones 7and 13 can be useful in these types of experiments.

Example 3 Use of an Anti-FMC63 Antibody for Purifying Macromolecules andCells

The antigen binding molecules disclosed herein are anti-idiotypicantigen binding molecules, and recognize an epitope on the anti-CD19scFv FMC63. An antigen binding molecule (e.g., an antibody) disclosedherein can thus be used to purify a molecule, such as the anti-CD19 scFvFMC63, macromolecule, polymer, cell, material, etc., displaying anepitope that is recognized by the antigen binding molecules disclosedherein.

In some embodiments, an antigen binding molecule disclosed herein (e.g.,Clones 7, 13, 14-1, 14-7, 15 and/or 17 and fragments thereof) can beattached to beads, attached to or associated with a resin, which can bedisposed in a column or other structure. A sample comprising a moleculecomprising all or a fragment of anti-CD19 scFv FMC63 can then becontacted with the beads, resin, etc to which the antigen bindingmolecule was attached or with which an antigen binding molecule wasassociated. This allows the formation of an association or bindingcomplex comprising the antigen binding molecule and the moleculecomprising all or a fragment of the anti-CD19 scFv FMC63. The beads orresin can then be washed with a suitable solution, such as a buffersolution (e.g., PBS, HEPES, MOPS, Tris, Tricine, etc) having a pHselected to maintain the stability of the molecule comprising all or afragment of the anti-CD19 scFv FMC63. The washing can remove unwantedand unbound components of the sample. Following the washing step, themolecule comprising all or a fragment of the anti-CD19 scFv FMC63 canthen be eluted from the antigen binding molecules using an elutionbuffer and conditions selected to disrupt any association or bindingcomplexes formed. Examples of suitable elution buffers include 0.1Mglycine, pH 2.5-3.0, and 0.1M citric acid, pH 3.0, 50-100 mMtriethylamine or triethanolamine, pH 11.5, 3.5-4.0M magnesium chloride,pH 7.0 in 10 mM Tris, 2-6M guanidine, and 2-8M urea. During the elutionstep, eluted molecules, cells and moieties of interest comprising all ora fragment of the anti-CD19 scFv FMC63 is collected, and purity can besubsequently checked by running a sample on an SDS polyacrylamide gel.

In another embodiment, an antigen binding molecule can be disposed insolution with any molecular entity displaying the epitope, and purifiedfrom a mixed population of molecules, cells, etc. and eluted from thebeads, resin, or free antibody by washing with 300-500 mM sodiumchloride or lowering the pH and neutralizing with 1 M Tris, forproteins, or phosphate buffer. Subsequently, dialysis can be used toreturn materials to desired buffer conditions.

In some embodiments, cells displaying a molecule comprising all or afragment of the anti-CD19 scFv FMC63 can be incubated with magneticbeads (e.g., DYNABEADS) with which an antigen binding molecule disclosedherein has been associated. Preferably the incubation is performed underconditions that both allow for the formation of bindingcomplexes/associations, such as under physiological conditions, in thepresence of a media selected for this purpose (e.g., RPMI-1640).

Cells bound by the beads (which will be presenting molecules comprisingthe anti-CD19 scFv FMC63) are then separated from cells not displaying amolecule comprising the anti-CD19 scFv FMC63 or fragment thereof. Insome embodiments, the beads can be washed with media, such as RPMI-1640supplemented with 10% FBS, in the presence of a magnet.

Selected cells, i.e., those presenting molecules that comprise theanti-CD19 scFv FMC63 can then be separated from the beads: First,selected cells are grown out in media. After growing out cells for 48hours, the magnetic beads can be separated from cells in solution anddiscarded, leaving a pure population of cells expressing desiredmolecule.

In an alternative embodiment, the beads are not magnetic, and in thisembodiment the above steps can also be followed and adapted to maintaincell integrity, but also to allow separation of bead-bound cells fromnon-bead bound cells.

In another alternative embodiment, an antigen binding molecule disclosedherein (e.g., Clones 7, 13, 14-1, 14-7, 15 and/or 17 and fragmentsthereof) can be His-tagged (i.e., labeled with a short polyhistidinesequence), thereby facilitating the separation of cells using a resincomprising a transition metal ion such as Ni²⁺, Co²⁺, Cu²⁺ or Zn²⁺,which are immobilized on the resin. The antigen binding molecules canthen be incubated with cells known or suspected to be expressing SEQ IDNO: 1 under conditions suitable for the formation of complexescomprising the cells and the antigen binding molecules. Following theincubation, the cells are contacted with the resin, which can bedisposed in a solid structure such as a welled plate, column or otherstructure. The antigen binding molecule-cell complexes can then beseparated from one another by washing with imidazole, which will be of ahigher concentration than any imidazole included in any solutions usedin the formation of the binding complexes. Eluted cells can then be spundown, washed in RPMI or other suitable media, and then resuspended inmedia.

Example 4 Activating CAR-Positive T Cells Using an Anti-FMC63 AntigenBinding Molecule

Also provided is a method of activating CAR-positive T cells presentinga molecule comprising a specific idiotope recognized by a specificantigen binding molecule (e.g., an antigen binding molecule thatrecognizes the anti-CD19 scFv FMC63 (SEQ ID NO: 1), such as thosedisclosed herein: Clone 7, 13, 14-1, 14-7, 15 and/or 17, and fragmentsthereof). This method may be adapted for any antibody recognizing aprotein of interest on a T-cell containing an activation domain, such asa chimeric antigen receptor (CAR) comprising SEQ ID NO: 1. Activationcan be achieved using plate-bound, bead-bound, polymer-bound, or otherform of the antibody that specifically recognizes an extracellularcomponent of the CAR or similar molecule.

In some embodiments, the method can be performed as follows: first, a12-well tissue culture treated plate is coated with 1 mL of a 1.5 μg/mLsolution of an anti-CD19 scFv FMC63 antigen binding molecule disclosedherein, which has been diluted in HBSS or other phosphate buffer, andplaced in an incubator at 37 C for 2 hours. Next, the plate is washedthree times with HBSS or other phosphate buffer having a suitable pH,ionic strength, etc. Continuing, CAR-positive T-cells in OpTmizer media(with supplements) or RPMI-1640 media with 10% FBS are added to thetissue culture treated plate. The cells are then grown at 37 C with 5%CO₂.

After 2 days, the cells are examined to determine any increase in thepercent CAR-positive cells. This determination can be made byidentifying any increase in the expression of any cell-surface and/orinternal markers, including, but not limited to 4-1BB, CD69, CD25, PD-1,and Ki-67.

Example 5 Generation of Humanized Sequences from Rabbit Antibodies

The Molecular Operating Environment (MOE) software developed by ChemicalComputing Group (CCG) can be used to generate alignments between therabbit antibody Clones 7, 13, 14-1, 14-7, 15 and 17 and pairs ofvariable light and heavy chains, VL and VH, respectively from twodatabases:

-   -   (1) The Abysis human database: a database of about 2000 known        human VL/VH sequence pairs from IMGT-LigM DB; and    -   (2) A human germline database: a database of germline sequences.

Humanized models show the best sequence alignments (highest identity toboth the VL and VH domains) with fewest gaps. The top 100 antibody pairsfrom each human database can be exported and clustered using kClust(Hauser, Mayer, & Soding, (2013) BMC Bioinformatics, 248). Tables for VLand VH sequences for each of the antibodies, can be constructed, withsequences from each of the two databases clustered at 90% and 95%.

Example 6 Flow Cytometry of Humanized FMC63 CAR

Humanized FMC63 CAR constructs were identified comprising conserved CDRloops (e.g., comprising amino acid sequences defined in Table C and D)and bind to CD19. Jurkat cells expressing FMC63 and humanized FMC63 CARconstructs were generated by transduction with lentivirus carrying therespective constructs. Monoclonal antibodies 14-1, 15, and 17-4 wereincubated with CAR-expressing Jurkat cells and detected with aFITC-conjugated goat anti-rabbit F(ab′)2 antibody (Thermo). An antibodyreactive against all of these CAR constructs was used as a control toverify expression (FIG. 22, top row). Clone 15 binds to the common aminoacids in the CDR loops of FMC63 and all the humanized constructs, butshows no reactivity against mock-transduced Jurkat cells. (FIG. 22).Secondary antibody only and mock-transduced controls show no backgroundstaining.

TABLE C CDRs for the light chain variable (VL) region of the humanizedFMC63 anti-CD19 SEQ SEQ SEQ Sequence ID ID ID (Convention) CDR1 NO CDR2NO CDR3 NO VL (Chothia) RASQDISKYL 74 HTSRLHS 75 QQGNTLPYT 76 NVL (Kabat) RASQDISKYL 74 HTSRLHS 75 QQGNTLPYT 76 N VL (IMGT) RASQDISKYL74 HTSRLHS 75 QQGNTLPYT 76 N

TABLE D CDRs for the heavy chain variable (VH) region of the humanizedFMC63 anti-CD19 SEQ SEQ SEQ Sequence ID ID ID (Convention) CDR1 NO CDR2NO CDR3 NO VH (Chothia) GVSLPDY 77 WGSET 78 HYYYGGSYAM 79 DY VH (Kabat)DYGVS 80 VIWGSETTYYNSAL 81 HYYYGGSYAM 79 KS DY VH (IMGT) GVSLPDYGV 82VIWGSETTYYNSAL 81 HYYYGGSYAM 79 S KS DY

What is claimed is:
 1. An isolated antigen binding molecule thatspecifically binds a molecule comprising SEQ ID NO: 1, the isolatedantigen binding molecule comprising: (a) a VH CDR1 region comprising theamino acid sequence of SEQ ID NO: 5; (b) a VH CDR2 region comprising theamino acid sequence of SEQ ID NO: 6; (c) a VH CDR3 region comprising theamino acid sequence of SEQ ID NO: 7; (d) a VL CDR1 region comprising theamino acid sequence of SEQ ID NO: 11; (e) a VL CDR2 region comprisingthe amino acid sequence of SEQ ID NO: 12; and (f) a VL CDR3 regioncomprising the amino acid sequence of SEQ ID NO:
 13. 2. The antigenbinding molecule of claim 1, wherein the antigen binding molecule isselected from the group consisting of an antibody, an scFv, a Fab, aFab′, a Fv, a F(ab′)₂, a mouse antibody, a rat antibody, a non-humanprimate antibody, a humanized antibody, a chimeric antibody, amonoclonal antibody, a polyclonal antibody, a recombinant antibody, anIgE antibody, an IgD antibody, an IgM antibody, an IgG1 antibody, anIgG1 antibody having at least one mutation in the hinge region, an IgG2antibody an IgG2 antibody having at least one mutation in the hingeregion, an IgG3 antibody, an IgG3 antibody having at least one mutationin the hinge region, an IgG4 antibody, an IgG4 antibody having at leastone mutation in the hinge region, an antibody comprising at least onenon-naturally occurring amino acid, and any combination thereof.
 3. Theantigen binding molecule of claim 2, further comprising a heavy chainvariable region (VH) sequence selected from the group consisting of SEQID NOs: 3 and
 15. 4. An antigen binding molecule, which comprises a VHamino acid sequence that is at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99%, or about 100% identical to a VH of an antigen bindingmolecule of claim
 3. 5. The antigen binding molecule of claim 2, furthercomprising a light chain variable region (VL) sequence selected from thegroup consisting of SEQ ID Nos: 9 and
 18. 6. An antigen bindingmolecule, which comprises a VL amino acid sequence that is at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, or about 100%identical to a VL of an antigen binding molecule of claim
 5. 7. Theantigen binding molecule of claim 2, wherein the antigen bindingmolecule comprises: (a) a VH comprising the amino acid sequence of SEQID NO: 3; and (b) a VL comprising the amino acid sequence of SEQ ID NO:9.
 8. The antigen binding molecule of claim 2, wherein the antigenbinding molecule comprises: (a) a VH comprising the amino acid sequenceof SEQ ID NO: 15; and (b) a VL comprising the amino acid sequence of SEQID NO:
 18. 9. The antigen binding molecule of claim 1, wherein theantigen binding molecule further comprises a detectable label.
 10. Theantigen binding molecule of claim 9, wherein the detectable label isselected from the group consisting of a fluorescent label, aphotochromic compound, a proteinaceous fluorescent label, a magneticlabel, a radiolabel, and a hapten.
 11. The antigen binding molecule ofclaim 10, wherein the fluorescent label is selected from the groupconsisting of an Atto dye, an Alexafluor dye, quantum dots,Hydroxycoumarin, Aminocouramin, Methoxycourmarin, Cascade Blue, PacificBlue, Pacific Orange Lucifer Yellow, NBD, R-Phycoerythrin (PE), PE-Cy5conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX,Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC,X-Rhodamine, Lissamine Rhocamine B, Texas Red, Allophycocyanin (APC),APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66Hmutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire,Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation),mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Cyan,Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65Lmutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1,TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1,Kusabira Orange, mOrange, Allophycocyanin (APC), mKO, TurboRFP,tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry,TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoeryhring(BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP),mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.
 12. A compositioncomprising the antigen binding molecule of claim 1.